Bakari AG
Department Of Medicine Ahmadu Bello University Teaching Hospital, Zaria
Lawal N; Akuyam SA ; Anaja PO
Department of Chemical Pathology Ahmadu Bello University Teaching Hospital, Zaria

All correspondence to: Lawal N nasiruacademy@gmail.com

ABSTRACT

Type 2 diabetic patients are at an increased risk of developing liver diseases owing to the nature of the disease and its inherent complications. Elevated serum activities of the liver enzymes are the most frequent indicators of liver disease. The purpose of this study was to determine the pattern of abnormal serum liver enzymes activities in type 2 diabetic individuals. The study comprised of 170 type 2 diabetic patients attending Medical Outpatients Department of Ahmadu Bello University Teaching Hospital, Zaria. Diabetes mellitus (DM) was confirmed according to the new diagnostic criteria based on 2 fasting or 2 random plasma glucose levels of more than 7.0 mmol/L and 11.1 mmol/L respectively. A concise history of the patients, physical examination and laboratory findings were recorded on a proforma. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using the kinetic method of IFCC. Serum gamma glutamyl transferase (GGT) activities were measured using the kinetic method of SZASZ. Serum alkaline phosphatase (ALP) activities were measured using the colorimetric method of King and Amstrong. The concentrations of serum FBG and RBG were measured using glucose oxidase method of Trinder.
One hundred and eighteen (69.4 %), 46 (27.1 %), 26 (15.3 %) and 51 (30.0 %) of patients had mild increases in serum levels of AST, ALT, GGT, and ALP respectively. In addition 42 (24.5 %) patients had both mild increases of AST-ALT and 10 (5.9%) had mild increases of all the liver enzymes activities. It can be concluded from the findings of the present study that there is chronic mild increases in serum liver enzymes activities in diabetic patients therefore, liver function tests (LFTs) be included into routine laboratory investigations of DM in Nigerian hospitals.

KEY WORDS: Diabetic, Serum, Liver Enzymes

INTRODUCTION

Diabetes mellitus (DM) is a systemic disease caused by absolute or relative deficiency of insulin and is manifested by disorders of carbohydrates, lipid and protein metabolism1. The prevalence of diabetes is high in patients who have liver disease such as non alcoholic fatty liver disease (NAFLD), chronic viral hepatitis, haemochromatosis alcoholic liver disease and cirrhosis. Similar studies have shown that DM plays a significant role in the initiation and progression of liver injury (Hickman IJ and MacDonald GA, 2007).
Onyemelukwe and Bakari (1998) observed that chronic liver disease was responsible for secondary diabetes mellitus in 15 cases (2% of total and 36 % of secondary diabetes mellitus). Of this number, 10 were secondary to liver cirrhosis, 3 with schistosomal liver fibrosis and 1 case each secondary to chronic active
hepatitis and chronic persistent hepatitis. All cases except Schistosomal liver fibrosis tested positive to serum hepatitis B surface antigen.
The hallmark of the disease is fasting hyperglycemia (WHO; Geneva, 1999) and studies have shown that liver plays a critical role in carbohydrate homeostasis and insulin degradation therefore, it is not surprising that it’s function may be affected by DM (Hanley et al. 2004). Association exists between DM and liver injury (Meltzer A and Everhart JE 1997). There are evidences have revealed that patients with type 2 DM have two times the risk of developing liver diseases than their healthy counterparts (Karen Barrow, 2005). Hsiao et al;2007 reported that alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyltransferase (GGT) were associated with insulin resistance (IR) as glycaemic status progresses in the impaired fasting glucose group.

Liver disorders among diabetics is similar to that of alcoholic liver disease including fatty liver (steatosis), steatohepatitis, fibrosis and cirrhosis. Elevated serum activities of the liver enzymes such as aspartate aminotransferase (AST), alanine aminotransferase(ALT), alkaline phosphatase (ALP) and gamma glutamyltrasferase(GGT) are the most frequent indicators of liver disease and occur in diabetics more frequently than in healthy individuals Hsiao et al; 2007.The aim of the present study was to determine the pattern of abnormal liver enzyme activities in type II diabetic patients in Zaria , Northern Nigeria.

MATERIALS AND METHODS
The study was conducted in Ahmadu Bello University Teaching Hospital (ABUTH), Zaria, Nigeria. This study was approved by the ethical committee of the ABUTH, Zaria in accordance with the declaration of Helsinki. A total of 170 diabetic patients attending Medical-outpatients Department (MOPD) and 80 apparently healthy individuals were studied. The criteria for diagnosis of type 2 DM was the American Diabetes Association Criteria (2004), fasting blood glucose of 7.0 mmol/L on two occasions or random blood glucose of 11.1 mmol/L with diabetic symptoms. The diabetic patients were provided with conventional diabetes care/control measures. At the MOPD, arrangement was made with the Physicians whereby subjects who satisfy the study inclusion criteria were selected. Informed consent for inclusion into the study was obtained from the subjects. The nature of the study was explained to the subjects by using an appropriate language. A full medical history was obtained from the subjects by the Physician followed by clinical examination. The findings were documented in the Proforma. Blood specimens were taken into plain tubes,
using sterile technique. The blood was centrifuged and the serum was carefully drawn into sample bottles and then stored frozen at -200C until the time for analysis. The samples were analyzed for plasma glycatedheamoglobin (GHbA1c) using the method of Triveli et al; 971 and Fasting Blood Glucose (FBG) as well as Random Blood Glucose (RBG) using the method of Trinder 1964..
Statistical analysis was performed using statistical package for social sciences (SPSS) for Windows, version 15.0. Data were presented as Mean±SEM. plasma glycated heamoglobin (GHbA1c) levels and serum Fasting Blood Glucose (FBG) as well as Random Blood Glucose (RBG) levels were compared with those of the apparently individuals using two tailed student t-test. A p-value of equal to or less than 0.05 (p=0.05) was considered as statistically significant.

RESULTS
The results of clinical parameters are presented in table I. The differences in weight and body mass index (BMI) between diabetic subjects and controls were statistically significant. Plasma GHbA1c and serum FBG as well as RBG in diabetic patients and controls are presented in table II. The mean values of plasma GHbA1c and serum FBG as well as RBG were significantly higher in diabetic patients than the control subjects (p<0.01).

The result of pattern of abnormal liver enzymes activities are presented in Table II. One hundred and eighteen (69.4 %) , 46 (27.1 %), 26 (15.3 %) and 51 (30.0 %) of patients had mild increases in serum levels of AST, ALT, GGT, and ALP respectively. Similarly, in the control subjects, 33 (41.3 %), 1 (1.3 %) 27 (36 %) 11 (13.8 %) individuals also had mild increase in serum levels of AST, ALT, GGT and ALP respectively.

DISCUSSION

The results obtained in the present study showed that the percentage prevalence of abnormal serum liver enzymes activities were significantly higher in diabetic patients than in control subjects. The finding of the present study was similar with those of Salmela et al; 1984 and Erbey et al; 2000 as seen in (table II). Even though there were variations in the values of percentage prevalence reported by the above mention authors as well as the present study however, the values were higher in diabetic patients than in control subjects.

The high percentage prevalence of elevated liver enzyme activities in type II diabetic patients seen in the present study suggest that the liver is diseased. It was reported that elevation of serum activities of the liver enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma glutamyltrasferase (GGT) are the most frequent indicators of liver disease (Monica et al 2005 and Meltzer A, Everhart JE 1997) therefore, the high percentage prevalence of elevated liver enzyme activities in type II diabetic patients as well as the higher mean level liver enzyme activities in type II DM than control subjects seen in the present study further confirm the abnormalities of liver function in type II DM. The hallmark of type 2 DM is hyperglycaemia and it leads to fat accumulation in the liver14. Fat accumulation is known to be directly toxic to hepatocytes . An elevation of liver enzymes such as ALT and GGT even within the normal range, reflects deposition of excess fat in the liver (Meybodi et al 2008 and Maryam et al 2008). This may be responsible for the increased serum activities of transaminases seen in the present study. Zachary, 2007stated that fat accumulation in the liver also stimulates the release of inflammatory cytokines such as tumor necrosis factor-α (TNF) and interleukine-6 (IL-6) which may contribute to hepatocellular injury. This might also result to increased serum activities of transaminases in the present study.

Hyperglycemia leads to myoinositol depletion in several tissues susceptible to diabetic complications (for example liver), although the exact mechanism is unclear. The fall in myoinositol levels in the cell membrane alters the functioning of Na+/K+ ATPase pump and the integrity of the cell membrane is impaired (Malnick et al; 2003). Clinical observations and experimental studies have shown that subtle changes of cell membrane are sufficient to allow passage of intracellular enzymes such as AST and ALT to the extracellular space with subsequent elevation of their levels in plasma (Malnick et al; 2003). This could be another reason for increased serum activities of transaminases in the present study.

Monica et al; 2005 reported that ALT, ALP and GGT were associated with prevalent type 2 DM and AST with prevalent IGT). NAFLD is a complication in 32%-78% of patients with type 2 DM and 50% of these patients may have NASH. Some studies demonstrated that hyperglycemia and hyperinsulinaemia can promote fatty infiltration of the liver (De Marco et al 1999).
CONCLUSION
It can be concluded from the findings of the present study that there were high prevalence abnormal liver enzyme activities in diabetic patients therefore, liver function tests (LFTs) should be included into routine laboratory investigations of DM in Nigerian hospitals.

ACKNOWLEDGMENT
We acknowledged the assistance of University Board of Research of Ahmadu Bello University, Zaria. We thank Dr IS Aliyu, Head of Chemical Pathology Department of Ahmadu Bello University Teaching Hospital (ABUTH), Zaria for his constant advice and encouragement during this study. We are also grateful to Dr Muazu Babura of the Department Medicine of ABUTH, Zaria for his assistance during the course of this work. We also thank Mal. MA Auwal and of the Department of Chemical Pathology ABUTH, Zaria for his assistance in analysis and other logistics.

Narendranath Epperla, Patrick Foy

Division of Hematology and Oncology, Medical College of Wisconsin, 9200 W Wisconsin Avenue, Milwaukee, WI, USA

Erika Peterson
Division of Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, WI, USA

Correspondences to: Narendranath Epperla · ·nepperla@mcw.edu

ABSTRACT
Background: Heterotaxy is a relatively uncommon congenital anomaly that is usually diagnosed incidentally on imaging studies in adults. We present an unusual case of venous thromboembolism in a 26 year old pregnant female with Heterotaxy syndrome.

Case presentation: A 26 year-old pregnant female at 13 weeks gestation suffered cardiac arrest with successful cardiac resuscitation and return of spontaneous circulation. The cardiac arrest was secondary to massive pulmonary embolism requiring thrombolytic therapy and stabilization of hemodynamics. She had extensive evaluation to determine the etiology for the pulmonary embolism and was noted to have an anatomic variation consistent with heterotaxy syndrome on imaging studies. After thrombolysis the patient was treated with UFH and then switched to enoxaparin without complication until 25 weeks of gestation when she experienced worsening abdominal pain with associated headaches, lightheadedness and elevated blood pressures needing elective induction of labor. The infant died shortly after delivery. The anticoagulation was continued for additional 3 months and she was subsequently placed on low dose aspirin to prevent recurrent venous thromboembolic episodes. She is currently stable on low dose aspirin and is into her third year after the venous thromboembolism without any recurrence.

Conclusion: To our knowledge, this is the first reported case of venous thromboembolism in pregnancy associated with heterotaxy syndrome. A discussion on pathophysiology of venous thromboembolism in pregnancy and heterotaxy syndrome has been undertaken along with treatment approach in such situations.

Keywords: Venous thromboembolism; Inferior vena cava; Low molecular weight heparin

BACKGROUND

Heterotaxy is an uncommon polymalformative syndrome characterized by congenital anomalies that arise from disorderly arrangement of asymmetric thoracic and abdominal viscera and blood vessels [1]. The estimated prevalence is approximately 1 in 10,000 live births. Morbidity and mortality rates are high approaching nearly 70 % due to major cardiac malformations (especially in heterotaxy with asplenia patients). Five to ten percent of patients with minor or no cardiac anomalies will survive to the adulthood. Some will remain asymptomatic and can be diagnosed incidentally on imaging studies. Heterotaxy has been reported to be associated with VTE and is felt to be related to abnormal lower extremity venous system. Infact in the last few years some authors considered it as a VTE risk factor. However VTE in pregnancy with heterotaxy syndrome has not been
reported.
Herein we present a case of 26 year-old pregnant female who suffered cardiac arrest related to massive pulmonary embolism requiring cardiac resuscitation. Imaging studies incidentally discovered anatomic variation consistent with heterotaxy syndrome. She was anticoagulated for 6 months with low molecular weight heparin (LMWH) and subsequently placed on low dose aspirin to prevent recurrent VTE. A detailed discussion of the pathophysiology of VTE in pregnancy and heterotaxy syndrome has been undertaken with a focus on treatment approach in such situations.

Case presentation
A 26 year old otherwise healthy, G1P0 female at 13 weeks gestation was admitted to medical intensive care unit after she experienced cardiac arrest at home. She had reported shortness of breath earlier that day and then collapsed.

During initial paramedic evaluation, she was pulseless and found to be in pulse less electrical activity (PEA). She was successfully resuscitated with return of spontaneous circulation (ROSC) in approximately 7 min and was brought to the hospital. Emergency department (ED) evaluation revealed the patient to be nonresponsive, and she was immediately intubated. Her exam was remarkable for cold and cyanotic extremities, without palpable distal pulses but strong femoral pulse. Shortly after her arrival to ED, the patient became pulse less again needing resuscitation and ROSC on 2 occasions.

Initial blood work was remarkable for severe metabolic acidosis (HCO3 of 10) and elevated troponin I (2.77 ng/ml, normal range 0–0.34 ng/ml). EKG showed right bundle branch block while bedside 2 D echocardiogram demonstrated right heart strain pattern. Bedside ultrasound (USG) showed no significant free fluid with live intrauterine gestation. Bilateral lower extremity compression venous dopplers of the entire venous system were negative for any evidence of deep venous thrombosis. She underwent computerized tomography (CT) of the chest which revealed a nearly occlusive thrombus in the bilateral lower lobar arteries extending into the segmental arteries (Fig. 1). The main pulmonary arterial trunk was noted to be enlarged with evidence of right heart strain (Fig. 2). Head CT scan was negative for any acute intracranial abnormalities. She was administered intravenous tissue plasminogen activator (TPA) (50 mg after ROSC the second time) in the ED with improvement in her hemodynamics. Hypothermia protocol was concurrently initiated. She was subsequently transferred to medical intensive care unit on unfractionated heparin (UFH), bicarbonate and norepinephrine drips. The patient was extubated two days following and was gradually weaned off blood pressure support. UFH drip was titrated based on aPTT and 6 days later she was transitioned to LMWH (dalteparin 10,000 units subcutaneously once daily in view of her insurance issues). She had aggressive supportive care and after a 2 week hospital stay, she was discharged to rehabilitation facility on Dalteparin. A month later dalteparin was changed to enoxaparin 55 mg (1 mg/kg) s/q twice daily (monitored with anti Xa levels).
At approximately 20 week’s gestation, the patient had a fetal ultrasound that revealed singleton gestation with massive hydrops, fetal contractures and ventriculomegaly/hydranencephaly. It was presumed that these findings were secondary to early hypoxic injury and she was counseled about poor prognosis. During her 25th week gestation, the patient noted worsening abdominal pain with associated headaches, lightheadedness and weakness. She had elevated blood pressures and was suspected to have mirror syndrome. Enoxaparin was discontinued 24 h prior to the planned procedure with initiation of UFH drip. The patient underwent elective induction of labor with palliative care services. The infant died shortly after delivery. The patient experienced increased vaginal bleeding secondary to retained products of conception and underwent dilatation and curettage with achievement of hemostasis. Enoxaparin at previous dosage (55 mg s/q twice daily) was restarted 24 h after the delivery without any major or untoward complication. Platelet count and anti-Xa levels were checked while the patient was on enoxaparin. Anti-Xa levels were in therapeutic range (0.8–1, goal 0.6–1.0 IU/ml) and platelet counts remained within normal limits.
In order to identify the cause of her VTE the patient had further work up including thrombophilia testing and CT chest, abdomen/pelvis. Thrombophilia testing revealed the patient to be wild type for Factor V and Prothrombin gene. Protein C and S antigen levels were normal. Antithrombin activity was initially low (71, normal range 75–125 % ACT) at the time of initial VTE but subsequently normalized (107). Antiphospholipid antibody testing including lupus anticoagulant, anti-cardiolipin antibodies and beta 2 glycoprotein I antibodies was negative. CT chest, abdomen and pelvis showed constellation of imaging findings compatible with heterotaxy syndrome. These included left-sided superior vena cava (SVC) draining into the left coronary sinus, hemiazygous vein drains into left SVC, absent azygous vein, absent (interrupted) inferior vena cava (IVC) below the hepatic IVC with persistent left IVC which drains into left coronary sinus, polysplenia, bilateral left lung and benign hepatic hemangioma (Fig. 3A-C).

The patient completed 6 month duration of anticoagulation with LMWH and was subsequently placed on ASA 81 mg. Prior to discontinuation of anticoagulation therapy the patient had CT pulmonary angiogram which showed normal diameter of the main pulmonary artery without any evidence of acute or chronic pulmonary emboli. She is currently stable on low dose ASA and is into her third year after the VTE without any recurrence. She has had no further pregnancies.

DISCUSSION
Heterotaxy or situs ambiguous refers to malposition and dysmorphism of viscera and blood vessels, often with indeterminate atrial arrangement. This abnormal arrangement of body organs is different from the orderly arrangement seen in situs solitus or situs inversus. Heterotaxy syndrome can be associated with either asplenia or polysplenia. Our patient had heterotaxy with polysplenia and hence will limit our discussion to this abnormality and hereon will be referred to as heterotaxy.
Heterotaxy implies that the patients have bilateral bilobed lungs, bilateral pulmonary atria, a centrally located liver, a stomach in indeterminate position, interrupted IVC with azygous continuation and multiple spleens. After polysplenia, the most frequent finding in heterotaxy is the hypoplasia of the IVC with absence of the intrahepatic segment and direct continuation with the azygous venous system. Heterotaxy commonly occurs as a sporadic condition but it can be inherited.
Varied timing of embryological causative factors during the development of fetus can explain the wide range of anomalies in the heterotaxy syndrome. Cardiac anomalies are mostly the result of abnormal embryological development around day 28 of embryogenesis, as it is during this period that connection between primitive heart and venous channels occurs. The failure of fusion of fetal lobules leads to individualization of multiple splenic nodules, which are located along the greater curvature of the stomach, as the spleen develops in the mesogastric region. IVC is a complex vascular structure that is developed during weeks 6–8 of embryogenesis. During this period three pairs of primitive venous channels (posterior cardinal, subcardinal and supracardinal veins) form the mature venous system (IVC) through a complex sequential process. Various malformations including partial or even complete absence of IVC can result from the failure in the developmental steps related to embryonic dysontogenesis that can affect separate segments or even the entire IVC. Thus mutations in genes that control left-right patterning and teratogenic exposures in early embryonic period underlie majority of heterotaxy cases.
The occurrence of DVT seems to be higher in patients with heterotaxy compared to the general population. It is postulated that stasis related to the abnormal venous drainage of the lower extremity venous system due to interrupted IVC, and increased platelet aggregation related to the anomalous drainage of the splenic venous system into the azygous system seems to be the possible culprits for heightened risk of pulmonary thromboembolism in these patients.
VTE is 5 times more frequent in pregnant women than in non-pregnant women of similar age. This is because pregnancy induces a state of venous stasis and
Fig. 3. A. CT scan of the chest with contrast.
Coronal section showing bilateral bilobed lungs
(yellow arrows depict the major fissure). B. CT
scan of the abdomen. The arrow points to multiple
splenic lobules suggestive of polysplenia.
C. CT scan of the abdomen. Coronal section
showing azygous continuation of inferior vena cava

hormonal and hematological changes leading to an increased risk for VTE. Venous stasis occurs from progesterone mediated increased venous distension in the first trimester and the compressive effect by the enlarging uterus on the common iliac vein in the late second and third trimesters. In addition there is an imbalance between the procoagulant (increased circulating levels of fibrinogen, von Willebrand factor, VII, VIII, IX, X and XII and increased generation of fibrin) and anticoagulant factors (decreased Protein S levels and fibrinolysis) through the pregnancy. Though pregnancy is considered to be a relative contraindication to systemic thrombolysis (recombinant TPA or streptokinase) in hemodynamically unstable patients with pulmonary embolism, the risk of complications for pregnant females treated with thrombolytic agents may be similar to that in the non-pregnant population.

In our case, the exact etiology for VTE is unclear. It may be related to pregnancy alone or mechanical factors from distorted venous system anatomy or a combination of both. Because the patient’s VTE occurred early in pregnancy, uterine enlargement is unlikely to have contributed to venous thrombosis. Effects of pregnancy in heterotaxy have been poorly described.

Challenges to the clinical management of our patient include the duration of anticoagulation and future pregnancy. Based on the 2012 American College of Chest Physicians (ACCP) guidelines, anticoagulation with LMWH is recommended for at least 3 months from the initial pulmonary embolism and 6 weeks postpartum. In our case, the patient was into her 3rd month of anticoagulation when she had induction of labor and delivery; hence the anticoagulation was continued for additional 3 months for a total of 6 months. Subsequently she was placed on low dose aspirin (81 mg) to prevent recurrent VTE (especially given her heterotaxy syndrome) based on the Warfarin and Aspirin (WARFASA) and Aspirin to Prevent Recurrent Venous Thromboembolism (ASPIRE) trials as well as the individual patient data analysis of WARFASA and ASPIRE trials performed by the INSPIRE Collaboration. Currently there is no evidence about the risk of VTE recurrence and anticoagulation duration in similar patients. According to the 2012 ACCP guidelines the decision regarding the duration of anticoagulation in a patient should be made after careful evaluation of both the risk of VTE recurrence and bleeding risk. Given the congenital prothrombotic risk factor (interrupted IVC related to heterotaxy syndrome), severity of the presentation and apparently low bleeding risk extended anticoagulation treatment with targeted oral anticoagulation maybe a suitable alternative to aspirin with periodic assessment of the bleeding risk.

Our patient had a massive pulmonary embolism with cardiac arrest that created a great sense of apprehension both in the patient and her family regarding future pregnancy. Unfortunately, there is paucity of data to either support or refute future pregnancy associated with her condition. Previous VTE alone is not a contraindication to future pregnancy provided anticoagulation is available. However patients with heterotaxy syndrome and VTE are undoubtedly at increased risk of VTE compared to other pregnant women. Hence future pregnancies in this patient group should be considered high-risk, and requires multi-disciplinary management. We recommend full intensity anticoagulation with LMWH (likely enoxaparin 1 mg/kg two times daily) with regular monitoring of anti-Xa levels prior to her pregnancy. It is important to measure anti-Xa levels 4 h after the last dose and be aware of the different targets based on which LMWH regimen is used (once-daily [goal 1.0–2.0 IU/mL] or twice-daily [goal 0.6–1 IU/mL]). In addition we recommend that there is a detailed discussion between the physician and the patient regarding the role of thrombolytic therapy in the event of recurrent VTE and possible termination of pregnancy in the worst case scenario.

CONCLUSION
To our knowledge this is the first reported case in the English literature that shows pregnancy associated VTE in a person with heterotaxy. After completion of anticoagulation therapy for the initial thrombotic event, the patient needs secondary prevention for VTE recurrence and low dose aspirin was the chosen treatment in this case. Although there are no published guidelines we believe that there are no contraindications for future pregnancy in this special group provided the patient is on anticoagulation prior to the pregnancy.

Enabulele, O.I.
Faculty of Life Sciences,Department of Microbiology, University of Benin Edo State, Nigeria.

Tchounga, K.S., Ukaji, D.C., Ajugwo’ A.O.
Department of Medical Microbiology/ Medical Mycology, Faculty of Medical Laboratory Science,
Madonna University Elele Campus, Rivers State,Nigeria
All correspondence to: ukajidamian@yahoo.com

ABSTRACT
Serotype and biotype of Vibrio cholerae isolated from stool samples collected from patients with acute diarrhea and water samples were determined during September to November 2004 cholera outbreak in Uzebba in Edo–State, Nigeria. A total of 137 stool samples and 50 water samples were investigated. The samples were subjected to standard recommended microbiological techniques and confirmation of isolates by seroagglutination using Vibrio cholerae polyvalent O1 and O139 antisera and monovalent Ogawa and Inaba antisera, while biotyping was carried out by agglutination test and sensitivity to polymyxin B. Out of 137 stool samples, 63((45.98%) were found to be positive for Vibrio cholerae serogroup O1, and of 63Vibrio cholerae O1, 60(95.24%) were Vibrio cholerae serogroups O1 serotype Inaba, biotype classical and 3(4.76%) were found to be Vibrio cholerae serogroup O1, serotype Ogawa, biotype classical. Only stream water samples yielded growth of Vibrio cholerae serogroup O1, with 3(23.7%) out of 13 stream water samples. Well and bore hole water samples yielded no growth of Vibrio cholerae. Out of three Vibrio cholerae O1 isolated from stream water samples, 2(66.67%) were found to be positive, for Vibrio cholerae serogroup O1, serotype Inaba and classical biotype, while 1(33.33%) was Vibrio cholerae serogroup O1, serotype Ogawa and El -Tor biotype. This study demonstrated that some Vibrio cholerae O1 serogroup O1 isolated from stream water and stool samples had the same serotypes and biotypes. Therefore, there is need to perform a strong epidemiological surveillance for emergence and distribution of Vibrio cholerae O1.

Key words: Vibrio cholerae O1, serotype, biotype, water supplies, stool samples.

INTRODUCTION
Cholera (frequently called Asiatic cholera or epidemic cholera) is an acute diarrheal illness caused by a Gram negative slightly curved rod Vibrio cholerae (Ryan and Ray, 2004). Cholera is a worldwide problem, especially in developing countries. It has been very rare in developed nations for hundred years; however the disease is common today in other parts of the world, including the India sub-Asian continent and sub-Sahara Africa. Cholera is a major health threat in poor nations that frequently results in mortality. Although more than 100 serogroups of Vibrio cholerae exist, only two cause human disease. Vibrio cholerae O1 of which there are two biotypes (Classical and EL Tor); classified into serotypes Ogawa and Inaba and rarely Hikojima and Vibrio cholerae O139, which emerged in 1992. Cholera world wide is usually characterized by painless diarrhea and vomitting.
In 1991, there were more than a million cases in Central and South Canada. Several new cases have since been reported in Louisiana and other Gulf Coast areas (Werdlow
et al., 2002). African countries have in recent years experienced more epidemics and (WHO, 1999). A total of 29321 cholera cases and 10586 deaths were reported to WHO in 1998, with Africa accounting for the largest part with 72% of the global total (WHO, 1999). An explosive outbreak of cholera occurred among Rwanda refugees in Goma. Democratic Republic of Congo involved about 70,000 cases and caused about 12,000 deaths in 1994 (Siddique et al., 1995). In 1995, West Africa reported 64% cholera cases and 61% cases of cholera deaths (WHO, 1999). Between July and November 2001, several outbreaks were reported in the region, including 897 cases with 47 deaths in Cote d’voire and 575 cases with 21 deaths in Kano (WHO, 2001). In 2004, another outbreak involving 1316 cases and 76 deaths were again reported in Kano, while Edo-State (Uzebba) witnessed an outbreak of cholera involving 300 reported cases with 50 deaths (WHO, 2004). The Onslaught of cholera in Africa continent continues unbeaten as another one was reported in Nigeria involving 400 cases in Jigawa State, resulting in 15 deaths in October 2009 (WHO, 2009).

Strategies for prevention and control of this infectious disease depend on understanding the origin, transmission and other characteristics associated with it epidemiology. Therefore, this study was carried out to determine the prevalent serogroup, serotypes and biotypes of Vibrio cholerae responsible for the cholera outbreak in Edo-State, (Uzebba) between September to November 2004.

Materials and Methods
Stool samples were collected from patients with acute diarrihea. During the outbreak, exclusion criteria included patients using antimicrobial agents within the previous two weeks to avoid cases of antibiotic associated diarrhea. Water samples were collected in clean containers containing alkaline peptone water (APW) from various sources of water supply (streams, boreholes and wells).

Sample Processing
Stool samples collected in plain containers were cultured directly onto Thiosulphate Citrate Bile Salt Sucrose (TCBS) agar plates and incubated at 37oc for 24 hours. Water samples inoculated in APW were incubated at 37oc for 4 to 6 hours, after the incubation period, from the inoculated APW, plating was done onto TCBS agar and the plates incubated at 37oc for 24 hours. The isolates were purified by sub-culturing single colonies on sterile Nutrient agar plates which were then incubated at 37oc for 24 hours. Following standard morphological and biochemical tests according to Buchanan and Gibbons (1974), characteristic colonies grown on the selective TCBS agar were then confirmed for identification. The series of biochemical tests commonly used to identify V. cholerae (Baumann and Schubert, 1984; West and Cowell, 1984) were originally designed for clinical samples in order to specifically detect pathogenic vibrios. The series of biochemical tests include: Gram staining, oxidase , glucose and L-arabinose , methyl red , voges-proskauer and ornithine tests were performed.

Serological Test
Serological identification of isolates was done using slide agglutination test (Sakazaki and Donovan, 1984; Shimada et al., 1994), using polyvalent Vibrio cholerae O1 and Vibrio cholerae O139 antisera and monovalent Ogawa and Inaba antisera.

Biotyping
Biotyping of Vibrio cholerae isolates was done by using agglutination with chicken red blood cells and polymixin in B (50 units) sensitivity test (WHO, 1987).

RESULTS
A total of 137 stool samples collected from hospitalized patients during cholera outbreak from September to November 2004 in Uzebba (Edo – State), and water samples 50(13 stream water, 20 borehole water and 17 well water samples) were studied.

Table 1 shows the frequency of isolation, serotyping and distribution of Vibrio cholerae. Isolated from stool and water samples in Uzebba. Out of 137 stool samples, 63(45.98%) were found positive for Vibrio cholerae O1 and 60(95, 24%) out of 63, belonged to Inaba serotype, while 3(4.76%) were Ogawa serotype. None of the samples yielded growth of Vibrio cholerae O139. Water samples were also analyzed bacteriologically, 3((23.07%) out of 13 water samples collected from streams yielded growth of Vibrio cholerae O1 and borehole and well water samples yielded no growth. Table 2 shows the distribution of Vibrio cholerae O1 biotype isolated from stool and water samples. All vibrio cholerae O1 from stool samples both Inaba and Ogawa serotypes were Classical biotype, two Vibrio cholerae O1 Inaba serotype isolated from stream water samples were Classical biotype, while only Vibrio cholerae O1 Ogawa isolated from stream water sample was EL Tor biotype.
DISCUSSION
Cholera continues to be an important pubic Health problem among many poorer and vulnerable communities despite the fact that bacteriology, epidemiology and public health aspects of the disease were described in detail over a centuary (Shears, 2001). In the study, we isolated and identified 66 Vibrio cholerae out of which 63 isolates were from stool samples and 3 isolates from water samples.It was observed after serological test that the isolates were Vibrio cholerae O1 and that there was no serogroup O139 isolates. These findings are in agreement with the report of Tamang et al.,( 2005) reported that Vibrio cholerae O1 were predominant in Nepal, also similar reports were presented by Urassa et al.,( 2000);Inaet al.,(2007) reported that during his study in Manhica District hospital Southern Mozambique, all isolates were Vibrio cholerae O1. During this study, it was noticed that two serotypes Inaba and Ogawa co-existed with Vibrio cholerae O1 serotype Inaba being the most predominant with 62(93.94%) while 4(6.06%) were Vibrio cholerae O1 Ogawa serotype; and these isolates were both from stool and water samples. Our findings are in accordance with reports of Mercy et al.,( 2004) who isolated Vibrio cholerae O1 in Ghana with Inaba serotype having the highest occurrence over Ogawa serotype. Similar report was presented by Shukla et al., ( 2006 ) who recorded an emerged predominance of Vibrio cholerae O1 serotype Inaba over Ogawa between 2004 – 2006 in East Delhi.

The co-existence of the two serotypes recorded during our study was not in conformity with Inacio et al., (2007) who reported that only Vibrio cholerae O1 serotype Ogawa was isolated in Manhica District hospital Southern Mozambique. Our findings also contradicted claims by Hossein et al., ( 2005) that after a six year study on Vibrio cholerae in South Eastern Tran, all Vibrio cholerae O1 isolated were Ogawa serotype. EL Tor was the predominant biotype causing outbreak up till 1998. But during our study in Uzebba, the Classical biotype emerged predominant that although that one of the isolates from water sample was EL Tor biotype. The high emergence of Classical biotype in Uzebba could be justified by the
reports of Rafi et al., ( 2004) also isolated Vibrio cholerae O1 Classical biotype between 2000 – 2001 in Rawalpindi. This high occurrence of Vibrio cholerae O1 Inaba and Classical biotype in our study is not in accordance with the reports of Bradley et al.,(1997), Jacques et al.,( 2002) and Iwanaga et al.,( 2004), who reported that most of the strains of Vibrio cholerae O1 isolated from Madagascar, Bangladesh, Tanzania, Zaire, Latin America, Southern and Eastern regions of India were Ogawa serotype and EL Tor biotype. Our study revealed that high percentage of Vibrio cholerae isolated from both stool and water samples were Vibrio cholerae O1 Inaba and Classical biotype, which is also contrary to the findings of Hossein, et al.,(2005) that all strains of Vibrio cholerae O1 isolated between July and September 1998 in Goa belonged to EL Tor biotype, 53(66%) of them being Ogawa serotype, while 21(26%) were Inaba serotype.

Some isolates obtained from stream water samples when serotype and biotype were reported as Vibrio cholerae O1 Inaba serotype Classical biotype same as some of the isolates obtained from patient’s stool samples during the outbreak. One could be tempted to say that by referring to the phenotypic and genotypic characteristics of the isolates, this stream water that has been one of the sources of water supply in Uzebba community must have played an important role in the spread of cholera outbreak in that region. We can also say that outbreak in Uzebba is defined by social and environmental factors.The importance of aquatic reservoir depends on sanitary conditions of the community(CEDECO,2010. Cholera is spread mainly through drinking fecal – contaminated water. . While contaminated water remains the major route for cholera transmission (Shapiro, et al., 1999) food and utensils are also important (Rabbani and Greenough, 1999), emphasizing the importance of hygiene within the house hold (Fotedal, 2001).

Effective food hygiene measures include cooking food thoroughly and eating it while still hot; preventing cooked foods from being contaminated through contact with raw food or drinking water is important (Seas and Gotuzzo, ensuring proper management of excreta to avoid contamination of other water sources were important measures to reduce cholera transmission.   Education of the population at risk regarding appropriate hygienic practice is always recommended. Identification of local customs that place people at risk is also important in order to eliminate such practices (Sears and Gotuzzo, 2000). A greater understanding of the pathogen, its biology, ecology, epidemiology and strategies for treatment and prevention are essential to guide policies and programmes for the control of cholera. . Adequate measures to improve hygiene and sanitation and supply of safe potable water are needed to prevent any future outbreak of cholera in Uzebba.

Atiegha C. Igoni M , Victor I.,
Department Of Medical Laboratory Sciences, College Of Health Technology Otuogidi-ogbia , Bayelsa State.
Ayaowei I.T.
Department Of Health Information Management, College Of Health Technology Otuogidi-ogbia , Bayelsa State.
Eseimokumo Me.
Department Of Pharmarcy Technician Studies, College Of Health Technology Otuogidi-ogbia , Bayelsa State.

All correspondence to: IGONI M ; Bayelsa State College of Health Technology: e mail; achristo40@gmail.com.

ABSTRACT
The treatment of tuberculosis has evolved from streptomycin to the DOTS strategy .Even as at that it is a serious challenge. This retrospective study was carried out to evaluate the effectiveness of the method. A total of sixty seven(67) Tuberculosis cases were evaluated at the Tuberculosis Treatment centre Nembe comprehensive Health Centre, Bayelsa state , it covered a period of four (4) years, from 2009 to 2012. Out of sixty seven cases that continued their treatment regiment to the end sixty four persons were cured and in only seven instances persons shows the ineffectiveness of the drugs by testing positive to the bacilli during chemotherapy. It is therefore recommended for community and family based treatment.

Key words: DOTS, Cured, treatment completed and treatment failure.

INTRODUCTION
Tuberculosis, a major health challenge and leading infectious killer is caused mainly in man by Mycobacterium tuberculosis. The bacilli enters the body by inhalation of droplets or dust particles containing it. It occurs as pulmonary tuberculosis or non- pulmonary tuberculosis. In pulmonary tuberculosis the pulmonary aveoli and surrounding lymph glands are lodged by the bacilli resulting in lesion characterized by acute inflammatory reactions with accumulation of fluid and white blood cells around the aveoli, while the non pulmonary tuberculosis includes the renal and urogenital tuberculosis, military tuberculosis and tuberculosis meningitis with varying symptoms1.
In sub-saharan Africa, cases of tuberculosis have increased dramatically, overwhelming control programs 2. This could be as a result of the fact that tuberculosis infection control interventions are not routinely implemented in contrast to what happens in high income countries with low prevalence of tuberculosis where infection control policy is routinely observed3. However the launch of directly observed treatment shortcourse (DOTS) in 1995 by the world health organization (WHO) has shown to be an effective intervention that will lead to reduced tuberculosis transmission and decreasing number of tuberculosis cases4,5. Also it is among the most cost-effective global health interventions available today6,7.
The DOTS strategy is based around a short-course treatment regimen for a minimum of six months of four drugs in combination (2 months of Isoniazide, Rifampicin ,Pyrazinamide and Ethambutol), a good management practice, sputum smear microscopy for diagnosis and the direct observation of doses to ensure adherence. Treatment success under DOTS concerns two outcomes- cured and treatment completed: “cured” if patient finish the treatment regimen with negative bacteriology at the end of the treatment whereas “treatment completed” refers to patients who have finished the regiment in full without showing evidence of treatment failure and without negative bacteriology. This treatment regiment however use to suffer from a number of drawbacks, with the combination of available drugs, the duration of treatment cannot be reduced below six months without a significance in relapses although re-infection with a different strain of mycobacterium tuberculosis can cause tuberculosis recurrence, which is considered to be an important measure of efficacy of tuberculosis treatment8,9. Also when treatment is given under sub-optimal conditions, regiments are associated with high rate of patients non-adherence specially in low income countries which harbor high burden of diseases10. The consequence of this is increased mortality and creation of clinic infections, drug resistant cases. The World Health Organization estimates in 2004, 424,203 cases of multidrug resistant tuberculosis globally among which 181,408 cases occurred in patients who had already been treated with standard (first line) therapy11.
This study was therefore carried out to determine the effectiveness of DOTS (Directly Observed Treatment Short course) on tuberculosis in the Nembe Local Government Area of Bayelsa state south-south Nigeria.

MATERIALS AND METHODS
Setting Comprehensive Health Centre Nembe, is situated in Nembe, the headquarters of Nembe local government area, about eighty kilometers south-east of Yenagoa the capital of Bayelsa State, Nigeria.

Subjects
The subjects of this report are those patients that reported to the TB treatment centre and those patients who were got through surveillance in the surrounding communities. A total of sixty seven positive cases were involved in the study. These cases were got over a four year period (from 2009 – 2012)

Laboratory analysis
Patients were given sterile wide mouthed plastic containers which they voided sputum into. Three specimens were collected from them over a two days period (spot, early morning and spot). Sputum analysis was done using the Ziehl-Neelson technique as described by cheesbrough12.

Method of drug administration
The strategy adopted here is the short course regiment that lasts for six months. Upon visit to the treatment center, Zeihl-Neelson(AFB) test is carried out. Treatment commences on patients who are newly diagnosed i.e. their sputum show smear positive. After the initial test, a second test (Zeihl- Neelson) is conducted upon revisit at the end of the second month of chemotherapy. The same thing applies
upon a second and third revisit which is at the end of the fifth and sixth months respectively.
For category 2 patients i.e. those who defaulted treatment or had a relapse, treatment is for seven months. Treatment on the intensive phase is for three months after which the second AFB test is done. The third and fourth AFB tests are done at the end of the fifth and seventh month of chemotherapy. Patients that absconded during treatment were not included in this work.

In 2009 eighteen new cases of sputum positives were recorded (six scanty and twelve +s). Upon first revisit i.e. after intensive phase, all eighteen persons were tested smear (AFB) negative. On second revisit the same feet was recorded. In the last revisit seventeen tested negative while one patient was AFB positive: table 1.

In 2010, seventeen persons were tested positive with varying degree of intensity of infection ( one scanty, eighty +s, four ++s and four +++s). sixteen persons tested smear negative at the end of the first revisit while person was tested positive. At the end of the fifth month of chemotherapy all fifteen persons tested smear negative (second revisit). After six months of chemotherapy one patient was tested smear positive while the other sixteen persons that continued their treatment to the end were tested negative. Conclusively, eleven persons were cured while one person was termed a “Treatment Failure”: table 2
Table 3 represents the summary of the 2011 treatment outcome. There were ten sputum smear positive cases in
2011. Eight of them were graded +, while one patient had 2+ and another 3+. Seven persons tested AFB negative upon the first revisit and one patients sputum was positive. In the second and third revisits, all eight patients had there AFB test negative. i.e. in 2011 eight persons were cured of tuberculosis in the treatment center.
Two scanty cases, twelve +s and eight cases of 2+s were recorded in the year 2012, twenty two cases in all. Three scanty cases were recorded in the first revisit stage, while nineteen tests were negative. In the second and third revisit all twenty two tests were smear negative. in 2012 twenty two persons were cured of tuberculosis in the treatment center. Table 4

Discussion
In this four years under study, the success of this method of treatment has been so tremendous. Out of sixty seven cases that continued their treatment regiment to the end sixty four persons were cured and in only seven instances persons shows the ineffectiveness of the drugs by testing positive to the bacilli during chemotherapy. When the result was subjected to the chi-square test, it shows that there is a significant difference between those that are cured of TB by the dots strategy in the five years under study and those that were not cured by the strategy p<0.05, (234.845, df 3, assymp. Sig (2-sided) 0.000

Conclusion:
Since with the DOTS strategy it is very difficult for patients to skip treatment, (which in most times is the cause of resistance), the researcher recommends that this strategy should be adopted, be it community based or family based)

Acknowledgment
We wish to express our heartfelt thanks to God almighty for his abundance grace to carry out this research and the strength to stand it all, without him nothing would be done. We also greatly appreciate the provost of the Bayelsa State College of Health technology, Dr Adias T. Charles for his inspiration and motivation. Also, the research and manpower development department of the College was involved with the funding. Finally we thank the staffs of the comprehensive health centre Nembe, Bayelsa State.

Saleh, M.I.A., Mabrouk, M.A., Mohammed, A., Isa., A.I., Alhassan, A.W.

1. Department of Human Physiology, Faculty of Medicine, Ahmadu Bello University, Zaria, Nigeria.
Musa, K.Y.,

1. Department of Pharmacognosy and Drug Development, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Nigeria.

Ayaowei IT
Department Of Health Information Management, College of Health Technology Otuogidi-ogbia , Bayelsa State.
Helmy, A.
1. Biotechnology Centre, Misr University for Science and Technology, Cairo, Egypt.

All correspondents to: M.I.A. Saleh, Department of Human Physiology, Faculty of Medicine, Ahmadu Bello University,
Zaria, Nigeria. E-mail: alhajisaleh@yahoo.com

ABSTRACT
Nigella sativahas been used for medicinal purposes for centuries, both as a herb and when the seeds are powdered or pressed into oil in Asia, Middle East and Africa. It has been traditionally used for a variety of conditions and treatments related to respiratory health, stomach, intestinal, kidney, liver, circulatory and immune system support, and for general well-being .This study was aimed at investigating the effect of the n-butanol seed fraction of this plant as it affects gastric mucosal integrity and basal gastric secretions using the indomethacin –induced model. Phytochemical screening revealed the presence of flavonoids, alkaloids,, saponins, glucocinolates amongst others, whereas acute toxicity studies revealed a median lethal dose above 5000mg/kg. The rats were grouped into 6 (n = 5), with the extract fraction administered at 50, 100 and 200mg/kg subcutaneously, followed by pyloric ligation with indomethacin and cimetidine used as the standard drug. For the mucosal integrity study, ulcer and preventive indices were analysed, while volume of gastric juice, titratable acidity,acid output and pepsin concentration were assessed for basal gastric secretions. The three experimental doses of the extract at 50,100 and 200mg/kg showed a dose –dependent decrease in both ulcer and preventive indices. It also showed a significant (p<0.05) decrease in volume of gastric juice, titratable acidity, acid output and pepsin concentration in dose-dependent manner with the three experimental doses administered with the highest reduction at the 200mg/kg dose. The results obtained suggest that this fraction down regulated all those parameters which might be attributed to the presence of the phytoconstituents present in this fraction. Therefore, the extract fraction of this plant possesses gastroprotective and antisecretory effects further explaining the folkloric use of this plant in the therapy of peptic ulcer disease.
KEYWORDS: Nigella sativa, gastroprotection, basal secretions, NSAIDs

Introduction:

Peptic ulcers are breakages or discontinuities that can occur in the mucosal epithelial lining of either the stomach, small intestine, large intestine or the Merkel’s diverticulum along the gastrointestinal tract (Falase and Akinkugbe, 2010). They are known to occur world wide (Cemek et al., 2010) and are major causes of morbidity and mortality (Chaturvedi et al., 2007). The pathophysiology of peptic ulcer has been centralized on an imbalance between aggressive and protective factors. Factors such as stress, cigarette smoking, nutritional deficiencies, inadequate dietary habits, hereditary predisposition and frequent ingestion of non-steroidal anti-inflammatory drugs (NSAIDs) all are known to increase
the gastric ulcer incidences (Klein et al.,2010).In developing countries, usually 50-90% of the populations are infected with Helicobacter pylori, which is the main organism responsible for majority of peptic ulcer cases, and children acquire the infection soon after being weaned. Many natural products and modern synthetic drugs have been used to treat the peptic ulcer disease, but so far a complete cure has not been achieved or discovered, and exploration of new anti-ulcer drugs has remained a field of active research (Bandyopadhyay et al.,2001).Although there are many products in the market for the treatment of gastric ulcers, including antacids, proton-pump inhibitors, anticholinergics and H2-receptor antagonists, most of these drugs produce several adverse reactions,such as hypersensitivity reactions, arrythmias, impotence, gynaecomastia, nephrotoxicity, and haemopoetic changes(Chang and Leung.,2002; Scholl et al.,2005).Development of tolerance and incidence of relapses and side-effects on clinical evaluation make their efficacy arguable, further promoting non-drug compliance to therapy (Santin et al., 2011). In addition, most of these medications are expensive, which further restricts their use (Santin et al., 2010).
This has been the basis for the development of new antiulcer drugs, which include herbal drugs (Altinkaynak et al., 2003). Herbs are used in many domains including medicine, nutrition, flavouring, beverages, dyeing, repellants, fragrances and cosmetics (Djeridane et al., 2006). The plant Nigella sativa has been used for medicinal purposes for centuries, both as a herb when pressed into oil in Asia, Middle East and Africa. It has been traditionally used for a variety of conditions and treatments related to respiratory health, stomach, intestinal, kidney, liver, circulatory and immune system support, and for general well-being. The seeds are used as carminative, aromatic, stimulant, diuretic, antihelminthic, galactagogue and diaphoretic(Gupta et al., 2009).The aim of the present study was to evaluate the effect of the n-butanol fraction of this plant seeds on gastric mucosal damage and secretions.
2.0 Materials and Methods:
2.1 Plant material:
Nigella sativadried seeds were obtained during the month of July, 2011 from Sabon-Gari market in Zaria. Botanical identification and authentication was done by Mr. U.A Gallah at the Herbarium section of the Department of Biological Sciences, Ahmadu Bello University, Zaria. A voucher herbarium specimen (No: 101201) was deposited at the herbarium for future references.

2.2 Extraction of the plant material:
Nigella sativadried seeds weighing about 2kg were crushed and pounded with pestle and mortar. The powder was extracted with aqueous ethanol (70%) in a Soxhlet Extractor, concentrated using rotaryevaporator at reduced pressure, suspended in methanol and partitioned with n-butanol to obtain the n-butanol (n-BuOH) fraction. The fraction was further concentrated in-vacuo and the residue obtained. The extract yielded about 80% of the residue.

2.3 Phytochemical screening of the fractions:
The preliminary analysis for the extract was conducted for the presence of flavonoids, alkaloids, saponins, steroids, glycosides, anthraquinones, resins, reducing sugars and other phytochemicals using standard procedures for analysis (Evans, 2002 and Harborne, 2007).

2.4 Acute toxicity studies:
Lethal Dose (LD50) determination was conducted using the method of Lorke (1983). In the initial phase, male rats were divided into three groups of 3 rats each, making a total of 9 rats. The rats were treated with the n-butanol fraction of the extract at doses of 10, 100 and 1000mg/kg subcutaneosly. Animals were observed for 24 hours and the number of death(s) or those that showed neurological signs were recorded. In the second phase, the animals were grouped into 4 groups of one rat each and treated with the fraction at appropriate doses subcutaneosly. The rats were observed for 4 h for deaths or neurological signs, and the final LD50 was calculated as the square root of the highest non-lethal
dose in which the animal survived multiplied by the lowest lethal dose in which the animal died.

2.5 Drugs and chemicals/reagents:
Cimetidine (Lek Pharma, Slovenia), Indomethacin (Liomethacin(R))(Cheisi, Egypt),
Thiopental Sodium (Abbott Laboratories, UK), Phenol Red (BDH Poole, England), Sodium Hydroxide (NaOH) (BDH Poole, England) for the preparation of 0.01N NaOH solution, Phosphate Buffered Saline (PBS),Casein Substrate Solution 1% (w/v) (Sigma-Aldrich, USA), Hydrochloric Acid (HCl) 0.1N(Sigma-Aldrich, USA), Trichloroacetic Acid Solution 6% w/v (Sigma-Aldrich, USA). All other chemicals and reagents were analytical grade.
2.6 Experimental animals:
A total of ninety adult male albino Wistar rats were used in this study. The animals were obtained from the Animal House, Faculty of Medicine, El-Kasr el-Ain, Cairo University, Egypt. Their weights ranged from 180 – 240g. They were maintainedunder a similar conditions of humidity, temperature and light/dark cycle respectively and each of the animal was kept in a single individual cage, with wide-meshed galvanized wire bottoms to decrease coprophagy as much as possible. The rats were given access to food and water ad libitum for two weeks to acclimatize, prior to the commencement of the experiment. The rats were treated in accordance to the internationally accepted principles of laboratory animal use and care. At the time of the experiment, all treatments were conducted between 9:00 and 10:00 (GMT+1) h to minimize variations in animal response due to circadian rhythm. The animals were divided into the following groups and subgroups for gastric mucosal damage and gastric secretion studies respectively.

2.7 Experimental design:
Group I: Study of gastric mucosal damage
Group IA; Normal saline (Negative Control)Five rats received normal saline (1ml/kg/rat S.C).
Group IB: Indomethacin-treated (Positive Control) Five rats received indomethacin (20mg/kg S.C) for the study of gastric mucosal damage.
Group IC: Cimetidine-treated Ten rats for the study of the effect of two different doses of cimetidine 50 and 100mg/kg S.C on gastric mucosal damage (5 rats for each dose)
.Ten rats for the study of effect of two different doses of cimetidine (50mg and 100mg/kg) S.C, given 30 minutes prior to indomethacin administration on gastric mucosal damage (5 rats for each dose).
Group ID: Nigella sativa extract treated Fifteen rats for the study of the effect of n-Butanol (BuOH) fraction, each at three different doses (50, 100 and 200mg/kg S.C),when given 30 minutes prior to indomethacin on gastric mucosal damage (5 rats for each dose).
Group II: Study of basal gastric secretion
Group IIA: Normal saline (Negative Control) Five rats received normal saline (2 ml/rat S.C).
Group IIB: Indomethacin-treated (Positive Control)Five rats received indomethacin (20 mg/kg S.C), followed by pyloric ligation for the study of basal gastric secretion.
Group IIC: Cimetidine-treated Ten rats for the study of the effect of two different doses of cimetidine, 50 and

100mg/kg S.C on basal gastric secretion (5 rats for each dose).
Ten rats for the study of the effect of cimetidine, 50 and 100mg/kg S.C, given 30 mins prior to indomethacin administration on basal gastric secretion (5 rats for each dose).Group IID: Nigella sativa-treated Fifteen rats for the study of the effect of n-Butanol (BuOH) fraction, each at three different doses (50, 100 and 200mg/kg S.C),when given 30 minutes prior to indomethacin on basal gastric secretion (5 rats for each dose).

2.8 Induction of gastric ulceration
After 48 hours of starvation, the animals were weighed and maintained in their individual cages. Then, indomethacin 20mg/kg was injected subcutaneously and the animals were then deprived of both food and water for 7 h (Urushidani et al., 1979). The animals were later sacrificed by decapitation (Satoh et al., 1983). Their stomachs were opened along the greater curvature, rinsed slowly with running water, then stretched out as much as possible by the use of pins on No.1 Whatman’s filter paper on a ceiling board.

2.9 Quantitative assessment of mucosal damage
The ulcerated areas in each stomach were measured with a transparent (mm) ruler scale, the result of each group were expressed as ulcer index (U.I) in mm of mean ulcer ± standard error of mean (Scepovic and Radmanovic, 1984).
The degree of ulceration was expressed as ulcerindex (U.I). It was calculated by multiplying ulcer score by 100 (Robert et al., 1968). Ulcer score for each group was calculated by dividing the total number of ulcers in each group by the total number of rats in that group (Robert et al., 1968).The percentage preventive index was calculated according to the method of Hano et al.(1976), which is expressed as:
Preventive Index (%) = (U.I. Indomethacin – U.I. Extract/drug plus) indomethacin x 100
U.I. Indomethacin

2.10 Collection of gastric secretion
The gastric juice was collected according to the technique of Shay et al. (1954) as modified by Levine (1965), where oesophageal ligation was avoided. The animals were fasted for 48 hrs to ensure complete emptying of the stomach, but allowed water ad libitum. Each animal was weighed at the end of the fasting period. Light anaesthesia was sodium thiopental 10 mg/kg intraperitoneal (Juliane et al., 2009), abdomen of each rat was opened via a midline incision and the stomach exteriorated. A pyloric ligature was made using a thread with care to avoid damage to the blood vessels or traction to the stomach. The abdomen was then closed by suture, cleaned thoroughly with distilled water or saline, and the animal was allowed to recover.
After 3 h, the rats were sacrificed by decapitation, abdomen of each of the animals were opened. The oesophagus was ligated, and the stomachs removed and washed with distilled water or saline. An opening along the greater curvature was made (Nwafor et al., 2000) and the gastric content drained into a graduated centrifuge tube, the volume noted, then centrifuged at 1,006 x g for 15 minutes.

2.11 Analysis of the gastric juice
Determination of each sample volume after centrifugation
The volume of 3 hours gastric secretion was measured after being subjected to centrifugation at 1,006 x g for 15 mins.
Determination of titratable acidity: A given volume of the gastric juice (1ml) was titrated against 0.01N NaOH. An end point of pH 7.0 as determined colorimetrically at 280nm by phenol red was used (Grossman, 1973; Davenport, 1977).The values were calculated as micro-equivalents per litre (Meq/L), which is equal to the number of millitres (ml) of 0.01N NaOH required to neutralize 1ml of gastric juice.
Titritable Acidity =
Volume of 0.01 N NaOH (mol) which neutralized 1ml of gastric juice
10
Determination of acid output: This was calculated by multiplying the volume (ml) of the gastric juice of each animal by the titritable acidity in that animal.
Determination of pepsin concentration: Pepsin concentration which is the major factor involved in the proteolytic activity of gastric secretion was determined in terms of the amount of protease enzymes produced after incubation of the substrate for 30 minutes with pepsin. It was determined by the spectrophotometric method devised by Jongensen (1954) and Hawk et al. (1960).
2.13 Statistical analysis
All data were expressed as Mean ± S.E.M (standard error of the mean) using SPSS Version 20. Statistical evaluation was done by analysis of variance (ANOVA) followed by post-hoc analysis by Duncan and Scheffe. Values of p<0.05 were considered significant (Microcal Software Inc., Northampton, USA).
3.0 Results:
The phytochemical screening revealed the presence of the following phytoconstituents as depicted in table I below:

The Phytochemical Analysis of N-Butanol Fraction of Nigella sativa L. seed extracts. Acute Toxicity Studies
The toxicity studies of the n-butanol seed extract of Nigella sativa in the first phase after being observed for 24hr, the rats did not show any signs and symptoms of toxicity or death. In the second phase, none of the rats produced any toxic symptoms or mortality up to the dose level of 5000mg/kg body weight, hence, they were considered safe for further pharmacological screening.

DISCUSSION AND CONCLUSION
To evaluate the gastroprotective effect of N-butanol fraction of Nigella sativa, the model of acute ulcer induced by a non-steroidal anti-inflammatory compound indomethacin was performed.
In this model, it was found that treatment with normal saline (2ml/rat) which was isotonic with the plasma as a control, showed no significant ulcer or lesion index with a preventive index of 97%. The group that received indomethacin 20mg/kg alone produced the highest lesion index when compared with all the treatment groups. There were gastric mucosal congestion, oedema, haemorrhage, lamina epithelial necrosis, leucocytic infilteration, blood vessels congestion with foci of necrotic tissues in the lesions. These results are consistent with previous studies that reported similar histopathological derangements and mucosal oxidative stress effects that involves weakening of gastric mucous, leading to formation of lesions in the gastric epithelium (Cyer, 2000; Chang and Leung, 2002; Ryo et al., 2006; Valcheva-Kuzmanova et al., 2007; El-Moselhy et al., 2009).
It was observed that the group that received 50 and 100mg/kg of the standard drug cimetidine alone, showed a significant decrease in gastric lesions (p<0.05) compared to the indomethacin 20mg/kg, cimetidine plus indomethacin group in the ulcer index with preventive indices of 73 and 79%, respectively. This corroborated with the studies of Marivane et al. (2011) who reported a decrease in lesion index on the gastric mucosa of rats treated with the H2-receptor antagonist cimetidine compared to indomethacin.
With regard to the effects of three different graded doses of the N-butanol fraction of Nigella sativa, it was found that treatment with the 50, 100 and 200mg/kg significantly reduced the lesion index compared with the control group (p<0.05) in a dose-dependent manner in this indomethacin-induced model. The highest dose of 200mg/kg N-butanol fraction produced the highest preventive index of 96% above that of the standard drug 50 and 100mg/kg cimetidine. A similar finding was reported by Marivane et al. (2011) that reported a significant reduction in lesion index, total injured area and the percentage injured area when extract of Brassica oleracea was used on indomethacin ulcer model.
Both Brassica oleracea and Nigella sativa are found to contain some flavonoids, especially quecertin and kacempferol mainly in glycosodic form and these are secondary metabolites that are widely distributed in nature with several biological activities including gastroprotective potentials (Martin et al., 1998). Croton urucurana with high flavonoid content also exhibited same mucosal cytoprotective potentials (Esmeraldino et al., 2005; Alves et al., 2008). The flavonoid content of this fraction had prevented and exerted a protective effect possibly by its inherent ability to scavenge free radicals, inhibit lipid peroxidation, increase mucous and prostaglandin contents of the gastric mucosa (Alanko et al., 1999).
Through phytochemical analysis of the fraction of N-butanol, apart from flavonoids, the presence of terpenoids, tannins, cardiac glycosides, steroids, saponins were detected amongst others. To further support and corroborate the possible fractions for the mucosal cytoprotection exhibited by this fraction of N. sativa containing flavonoids are the studies by Alcaraz and Hoult, 1985 that flavonoids increase mucosal prostaglandin content, inhibit histidine decarboxylase thereby decreasing histamine secretion (Bromer and Landry, 1985).
The presence of saponins in the fractions of N. sativa to improve on mucosal integrity has been reported in several other studies where plants containing saponins have been shown to possess antiulcer activity in several experimental ulcer models. Among these, saponins isolated from the rhizome of Panax japonicus and the fruit of Kochia scoparia (which contain approximately 20% of saponins) have been demonstrated to possess gastroprotective properties (Matsuda et al., 2003) in conformity with this study. The protective activities of all these saponins are not due to inhibition of gastric acid secretion, but probably due to activation of mucous membrane protective factors (Borreli and Izzo, 2000). Moreover, several plants containing high amount of saponins have been shown to possess antiulcer activity in several experimental bioassays, probably acting as an activator of mucus membrane stabilizing factors (Morikawa et al., 2006). Similarly, presence of tannins, terpenoids in N. sativa fractions further validate the cytoprotective property in the gastric mucosa observed in our study as reported by Al-Rehaily et al. (2002), where several saponins, tannins, terpenoids were found to possess gastroprotective properties. Additionally, Terpenoids are a widespread class of secondary compounds with several pharmacological activities, including anti-inflammatory and antiulcer activities (Arrietta et al., 2003). Plant extracts of Eleagnus angutifolia, Hibiscus esculentus, Papaver rhoeas, Phlomis grandiflora, Rosa canina all with a high flavonoid and saponin contents as reported by Ilhan et al. (2003) showed potent in vivo gastroprotective activity similar to that of Nigella sativa.
The interference of the N-butanol fraction of the extract using same model was also evaluated on parameters of basal gastric secretion. This method is an important procedure that reveals the possible changes of gastric secretory physiological parameters relating to volume of gastric secretion, titratable acidity, acid output relating to pH and the most important proteolytic enzyme in the stomach pepsin. The findings suggest that this fraction interfered with these major basal secretory indices of gastric juice.
Considering that the volume of gastric juice which is mainly acidic encompasses mucus, hydrochloric acid, pepsinogen, bicarbonates, intrinsic factor and protein plays a vital role in the aetiopathogenesis of gastric mucosal integrity, it is plausible to consider this fraction as a putative cytoprotective agent. This assumption was made based on the observation that the different quantities or volume of gastric juice obtained in this study showed a general inhibitory pattern with regard to its production in the stomach in this fraction evaluated. There was a significant reduction in the volume of gastric juice at the 100 and 200mg/kg extract treated groups when compared with the control. These results are in agreement with the studies of Muriel et al., (2008), who reported a significant decrease in volume of gastric juice on a similar ulcer model after using Green propolis.
With regard to the titratable acidity, there was no significant difference in all the three doses of N-butanol evaluated when compared with the control. Acid output significantly decreased in a dose related manner when compared with control. This decrease in stomach acidity facilitated the healing of gastric ulcers, because exposing the mucosa to high concentrations of acid favours mucosal epithelial damage (Laine et al., 2008). Contact between stomach acid and the mast cells of the submucosa and lamina propria causes mast cell degranulation and the release of histamine, which stimulates hydrochloric acid secretion by parietal cells and produces inflammation and acute oedema at the site of contact (Rodrigues et al., 2008). Overall, concentration of hydrogen ions in the gastric juice decreases reflective of high pH, further aggravating the aggressive factors (Lullmann et al., 2000). Consistently, hyperacidity is known to result due to uncontrolled hypersecretion of hydrochloric acid from parietal cells of gastric mucosa through the proton pump H+-K+ ATPase (Kishor et al., 2007). The results of 50mg/kg N-butanol was insignificant. Proteolytic activity as pepsin concentration significantly decreases (p<0.05) in a dose-dependent manner compared to control. A similar finding was reported by Halter et al. (1988) and Hatazawa et al. (2006).
In all the cimetidine treated groups, the volume of gastric juice, titratable acidity and pepsin concentration all significantly reduced compared to control. Gastric acid decimation by cimetidine has been attributed to its ability to antagonize the binding of histamine to the H2 receptor on the parietal cell membrane (Banji et al., 2010).The reported results have validated the folkloric use of N. sativa in the therapy of peptic ulcer disease. N. sativa offers protection against NSAIDs-induced gastric ulceration and down-regulate basal acid secretions to promote mucosal cytoprotection. The presence of phytoconstituents in this medicinal plant might be responsible for those pharmacological actions. In this context, extracts and active principles from plants could serve as leads for the development of new drugs. Therefore, this plant specie(s) have a great potential to be used as a gastroprotective agent in combination with others or alone.

Dangana .A,

Hematology Laboratory, University of Abuja Teaching Hospital, Abuja
Ishaku .H, Victoria .I,

Histology Laboratory, University of Abuja Teaching Hospital, Abuja
Christy .C.F,

College of Health Sciences, Pathology Department, University of Abuja
Egenti B.N

College of Health Sciences, Community Medicine Department, University of Abuja

All correspondence to: isalemit@yahoo.co.uk

ABSTRACT
Breast Cancer is the most common non-communicable disease and non-cutaneous malignancy. it’s a rare disease among men which account for less than 1% of all cancers in men. We examined the trends in the prevalence rate of breast cancer in men among tissues submitted to histopathology laboratory university of Abuja Teaching Hospital. A total of 544 data collected consisting of men between the age 17-86years with the mean aged group of 56years and was analysed using Epi-Info version 6.1. it was found that the prevalence of breast cancer among men was 4(2.6%), fibroadenoma197(36.8%),fibrocytic disease 120(22.4%), granulomatouse mastitis 14(2.6%)lactating adenoma 11(2.1%),sclerosing adenosis 8(1.5%), the highest prevalence rate was found between the aged group of 39-48years(50%) followed by 39-48years(25%) and 79-88years (25%) respectively.

KEYWORD: Breast Cancer, Male.

INTRODUCTION

Cancer is a term used for diseases in which abnormal cells proliferate without control.Breast cancer in men is a rare disease that accounts for less than 1% of all cancers in men and less than 1% of all diagnosed breast cancers,Magno et al,2009It is a diagnosis for which optimal management is not clearly established and treatment guidelines are scarce. The medical literature regarding breast cancer in men consists mainly of case-control and retrospective studies, and there are no randomised prospective data for this disease. Recent emphasis therefore has been placed on extrapolating data derived from studies of breast cancer in women and using those data as a benchmark for treating men—what’s good for the goose is good for the gander.Most types of cancer cells eventually form a lump; growth or mass called a tumor, and are named after the part of the body where the tumor originates. Breast cancer begins in breast tissue, which is made up of glands for milk production, called lobules, and the ducts that connect the lobules to the nipple. The remainder of the breast is made up of fatty, connective, and lymphatic tissue.Edge, et al,2010 Breast Cancer constitutes a major publichealth issue globallywith over 1 millionnewcases diagnosed annually, resulting in over 400,000annualdeath. Veronesi et al,2005, Omaret al,2013.There is an international/geographical variation in the incidence of Breast Cancer. Incidence rates are higher in the undeveloped countries than in the developing countries. Parkin ,et al,2005, Pages et al,2001. In Africa, Breast Cancer has overtaken cervical cancer as the commonest malignancy affecting women and the incidence rates appear to be rising. Vorobiofet al,2001, Omar et al,2013.In Nigeria breast cancer has increased from 116 per 100,000 in 2001. Adebamowo and, Ajayi. 2000. The activities of breast cancer society in some parts of the globe regarding cancer control and prevention may also be responsible for the better quality of live for victims of cancer in those countries Braunstein, 1993,Stratton et al,1997. Breast Cancers in developing countries are diagnosed when they are at advanced stages, which may be responsible for the higher mortality. The cost of treating Breast cancer in the tropics is prohibitive for most breast cancer victims; this is largely due to poverty and low per capita income as most people live below $1 per day, Thomaset al,1992, Althuiset al,1997.Cancers of cervix and breast have become preventable and have been well controlled In developed nations. Health education to increase awareness on breast self examination, may also contribute to early detection and prevention of invasive breast cancer,Adebamowo and, Ajayi ,2000. Agbo,et al,2013. The aim of this work is to determine the epidemiology and prevalence of breast cancer in menamong breast tissues submitted to histolopathology of university of Abuja Teaching Hospital, and also to determine the prevalence and proportion of breast cancer amongst the age group.

This is a retrospective study comprises of 544 samples of breast tissue submitted to the histopathology laboratory of university of Abuja Teaching Hospital Gwagwalada Abuja Nigeria between 2010-2014.
Inclusion: Samples that have patient’s detail such as age, sex and diagnosiswere used
Exclusion: samples without age, sex ,and all females were excluded from the study.
Statistical analysis: Result of data were captured in excel and then analyzed using epi-info version 6.1

Methodology
Histological examinations
Breast tissues received registered, grossed and was processed in automatic tissue processor containing 12
beakers, The tissue was processed by allowing the tissue to passed through the following stages Fixation, Dehydration,Clearing,Impregnation,Embedding,Sectioning,Staining,Mountingthe tissues were passed from beaker 1,2,3 contains formalin where the tissues was allowed to stay for 30min each, beaker 4,5,6 contains alcohol and was allowed to stay for 1hour each, beaker 7,8,9 10 contains xylene and was allowed to stay for 1hr each while the last 2 beakers which is 11,and 12 contains wax and was allowed to stay in beaker 11 for 2hours (infiltration) and 12 for 2hours (impregnation).

Staining procedures for Haematoxylin and Eosin
Princples.
The haematoxylin is a basic dye and has affinity for the nucleus DNA & RNA which is acidic,the orange G6 has affinity for the acidophilic cells of the cytoplasm of superficial cells while the eosin azure has affinity for the cytoplasm of intermediate, parabasal and basal cells.

Methods
The smears was remove from fixatives and rinsed in descending grades of alcohol (80,70,50%),for 8secs each Stain in Harris alum-Haematoxylin for 4mins.
Wash in tap water for 1-2 mins, and was differentiated in 1% acid alcohol briefly.
It was then wash and blue in tap water for 3-5mins, and then transferred to two changes of 95% alcohol for a few seconds each.Stain in OG6 for 2minutes, Rinsed in two changes of 95% alcohol, and Stain in Eosin azure for 2-4mins.
It was rinsed in two changes of 95% alcohol .complete dehydration in absolute alcohol and cleared in xyleneand was mounted in DPX and examined to study the architecture of the tissues.
Any tissue examined and aberrations in the structural integrity of the tissues seen and a case of malignancy established, immunohistochemistry employed to profile the markers.

Principles of Immunohistochemistry
Immunohistochemistry (IHC) is a wide-used biological technique that combines anatomy, physiology, immunology and biochemistry. Developed from the antigen-antibody binding reaction, immunohistochemistry can be considered as a method that visualizes distribution and localization of specific antigen or cellular components in separated tissues, or tissue sections. Compared to other bio-techniques that are based on the antigen-antibody reaction such as immunoprecipitation, or western-blot, immunohistochemistry provides in situ information which promises a more convincing experimental result.
Major components in a complete immunohistochemistry experiment:
1) Primary antibody binds to specific antigen;
2) The antibody-antigen complex is formed by incubation with a secondary, enzyme-conjugated, antibody;
3) With presence of substrate and chromogen, the enzyme catalyzes to generate colored deposits at the sites of antibody-antigen binding.

Protocol

Prepare formalin-fixed, paraffin-embedded tissue sections (Step 1-8):
1. The freshly dissected tissue was fixed (<3mm thick) with 2% paraformaldehyde from 1h to overnight at room temperature.
2. The tissue was rinsed in running tap water for 5 min.
3. The tissue was then dehydrated through 70%, 80%, 95% alcohol, 5 min each, followed with 3 times of 100% alcohol, 5 min each.
4. The tissue was cleared in xylene for 2 times, 5 min each.
5. The tissue was immersed in paraffin for 3 times, 5 min each.

6. And Embeded in a paraffin block. The paraffin tissue block was then stored at room temperature for years.
7. The paraffin-embedded tissue block was section at 5-8 µm thickness on a microtome and floated in a 40°C water bath containing distilled water.
8. The sections were transferred onto glass slides suitable for immunohistochemistry. The slides were allowed to dry overnight and it was stored at room temperature until ready for use.
Immunostain formalin-fixed, paraffin-embedded tissue sections (Step 9-29):
9. The slides were Deparaffinizein xylene for 2 times, 5 min each.
10. The slides were transferred to 100% alcohol, for 2 times, 3 min each, and then transfer once through 95%, 70% and 50% alcohols respectively for 3 min each.
11. The endogenous peroxidase activity were blocked by incubating sections in 3% H2O2 solution in methanol at room temperature for 10 min to block endogenous peroxidase activity.
12. It was rinsed with PBS for 2 times, 5 min each.
13. The blocking buffer was drained from the slides.
14. 100 µl of diluted primary antibody was apply appropriately (in antibody dilution buffer, e.g. 0.5% bovine serum albumin in PBS) to the sections on the slides and incubate in a humidified chamber at room temperature for 1 h.
15. The slides were then washed with PBS for 2 times, 5 min each.
16. 100 µl of diluted biotinylated secondary antibody was apply appropriately (using the antibody dilution buffer) to the sections on the slides and incubate in a humidified chamber at room temperature for 30 min.
17. The slides were washed with PBS for 2 times, 5 min each.
18. 100 µl diluted Sav-HRP conjugates was apply appropriately (using the antibody dilution buffer) to the sections on the slides and incubate in a humidified chamber at room temperature for 30 min (keep protected from light).
19. The slides were washed with PBS for 2 times, 5 min each.
20. 100 µl DAB substrate solution was applied (freshly made just before use: 0.05% DAB – 0.015% H2O2 in PBS) to the sections on the slides to reveal the color of antibody staining.The color was allowed to development for < 5 min until the desired color intensity is reached. (Caution: DAB is a suspect carcinogen. Handle with care. Wear gloves, lab coat and eye protection.)
21. The slides were washed with PBS for 3 times, 2 min each.
22. The slides were Counterstain by immersing in Hematoxylin for 1-2 min.
23. The slides were rinsed in running tap water for 10 min.
24. Theslides were dehydrated through 4 times of alcohol (95%, 95%, 100% and 100%), 5 min each.
25. The slides were cleared in 3 times of xylene and coverslip using mounting solution. The mounted slides were stored at room temperature permanently.
26. The color of the antibody staining in the tissue sections were then observe under microscopy.

RESULT
Out of 544 data collected for breast tissues, 19 (3.6%) were males, Breast cancer showed a prevalence rate of 15

DISCUSSION
Of the 544 breast tissues submitted to histopathology laboratory of University of Abuja Teaching Hospital Gwagwalada, male study subjects shows a breast cancer prevalence rate of 4(2.6%) and the age range of the study subject 7-86,with the highest prevalence rate at 49–58yrs,(50%) followed by 39-48yrs and 79-88(25% and 25%) respectively which is agreement with a study carried out by (Kidman et al., 2000) in Jos with the age ranging from 12 -85 years which shows that Male breast cancer rate was 2% and also with 2.5% recorded in Benin by (Okobiaet al., 2001) and 1.47% recorded in Nnewi by (Anyanwu, 2000) respectively.
This result is not in agreement with the results obtained from Zaria (Hassan et al., 1995) and Jos which recorded 8.6% and 9% respectively. Also different from results obtained in Tanzania and Zambia which shows a prevalence rate of 6% and 15% respectively according to(Singgal et al, 2006) and (Ihekwaba, 1994). The high incidence of male breast cancer in Nigeria and Africa compare to Western countries have been attributed to hyperestrogenism due to endemic liver infections in Africa and environmental changes and lifestyle (Pere et al., 2007).
The definite etiology of male breast cancer is idiopathic just like other cancers. Factors such as alteration of hormonal milieus, family history and genetic alterations are known to affects its occurrence.Various studies have also shown that conditions that alter the estrogen-testosterone ratio in males predispose to breast cancer (Balleriniet al, 1990 and Casagrandeet al, 1988). Among these conditions, the strongest association is with Klinefelter Syndrome. Males with this condition have a fifty times increased risk and account for 3% of all breast cancer (Hultbornet al, 1997). Any condition associated with increased estrogen levels like cirrhosis and exogenous administration of estrogen (either in transsexuals or as therapy for prostate cancer) have been implicated as causative factors (Symmers, 1968, and Pritchard et al., 1988).
Androgen deficiency due to testicular disease like mumps, undescended testis, or testicular atrophy has been linked to the occurrence of breast cancer in men (Thomas et al., 1992) and (Mabuchi etal., 1985). Occupational exposure to heat and electromagnetic radiation, causing testicular damage and further leading to the development of male breast cancer have been postulated (Stenlund and Floderus, 1997) and (Pages et al., 2001). Hereditary breast cancers are known to occur in males.
Studies have found that gremlin mutations in BRCA1 and BRCA2 account most for this. (Stratonet al, 1997).Gynaecomastia has also been implicated as a risk factor (Braunstein, 1993).
The peak level incidence among Male was between 40 -86 years with mean age of 57 years which is similar to other results obtained in Nnewi, Nigeria(Anyanwu,2000) with a mean age of 60yrs and 66yrs obtained in Spain respectively (Ihekwaba,1994). KaiyumarContractor,et al,2008 also found average age mean of diagnosis to be 60yrs which is similar to the mean age of this study.
The burden of breast cancer among Nigerian women is becoming overwhelming, this may be due to the cultural practice in this country where most women seek alternative treatment and healing (ranging from herbal to religious help believing in the say that “It never my portion” or people attacking them in their villages and poverty rather than coming to die in the hospital. This partially explains why most women present with advance disease as our figure has shown (97.6%) thereby leaving them only the palliative option of treatment. Aggressive awareness campaign is the only way to change these attitudes.

CONCLUSION
The rise in breast cancer cases in this study population is an indication of inadequate or ineffective control measures to curtail the disease or due to diversion of global attention to HIV/AIDS and Tuberculosis in the country. Therefore, there is urgent need to step up activities through non-governmental agency to promote advocacy, national policy on training of personnel for clinical and self-breast examination, and nationwide screening program (Mammography) in order to enhance early detection, control the upward trends and reduce the mortality rate of breast cancer .Breast cancer aggressive awareness campaign should be increased to allow earlier detection so as to reduce the mortality and morbidity rate.

Saleh, M.I.A, Isa., A.I, Mabrouk, M. A.,  Mohammed, A., Alhassan, A.W

Department of Human Physiology, Faculty of Medicine, Ahmadu Bello University, Zaria, Nigeria.
Heba, D.
Animal Science Unit, El-Kasr el-Ain, Cairo University, Cairo, Egypt.
Musa, K.Y.

Department of Pharmacognosy and Drug Development, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Nigeria

All correspondence to: alhajisaleh@yahoo.com

ABSTRACT
Nigella sativaL. is one of the most extensively studied plants both phytochemically and pharmacologically.It is an annual flowering plant, native to Southwest Asia and indigenous to the Mediterranean region, Middle-East, but now widely found in India.In the UnaniTibb system of medicine, N. sativa is regarded as a valuable remedy for a number of diseases including gastric diseases. The present study was carried out to investigate the gastroprotective and antioxidant effect of ETAC fraction of Nigella sativa L. (Family; Ranunculaceae) seed extract on indomethacin-induced gastric ulcer model in adult male albino Wistar rats with the fraction of the extract used at doses of 50, 100 and 200 mg/kg using cimetidine as standard drug. Phytochemical screening of the fraction revealed significance and variations in the presence of flavonoids, alkaloids, tannins, saponins, steroids/triterpenes, glycosides/glucosinolates and free anthraquinones in the fraction, while acute toxicity studies revealed a lethal dose (LD50) above 5000 mg/kg. The extract was administered subcutaneously thirty minutes prior to indomethacin (20 mg/kg). The various parameters studied were malondialdehyde, superoxide dismutase and catalase activities. Pretreatment with the fraction at doses of 50, 100 and 200 mg/kg with the 50 and 100mg/kg doses led to a decrease, though not significant in catalase(CAT) and superoxide dismutase (SOD) activities,while malondialdehyde (MDA) concentration significantly increased (P<0.05)with regard to the extract treated groups. This inhibition of lipid peroxidation and the generation of MDA and related substances from lipids that react with thiobarbituric acid was found to be inhibited by this extract. The inhibition was found to increase when the concentration of the extract was increased to 200mg/kg. Thus, it appears that the anti-oxidant property of this extract could counteract oxidative damage caused by NSAIDs. In conclusion, the gastroprotective properties of this fraction evaluated may be attributed to its reducing effect on oxidative damage and neutrophil infilteration in tissues due to the presence of phytochemicals like flavonoids, alkaloids and tannins, amongst others, present in the seed extracts with various antioxidant and other biological activities.

Keywords: Nigella sativa, Free Radicals, Antioxidant, NSAID’s,

1.0 INTRODUCTION

Peptic ulcer disease is one of the most common gastrointestinal disorders which causes a high rate of morbidity and at times even mortality particularly in the developing world (Falk, 2001).The pathophysiology of these disorders has focused on an imbalance between aggressive and protective factors in the stomach such as acid-pepsin secretion, mucosal barrier , mucus secretion , blood flow, cellular regeneration, prostaglandins, and epidermal growth factors.Stress, smoking, nutritional deficiencies, and ingestion of non-steroidal anti-inflammatory drugs are all factors which increases the gastric hyperacidity and gastroduodenal ulcer incidences (Lima et al., 2006;Jainu and Devi, 2006).An imbalance between antioxidants and reactive oxygen species results in oxidative stress, leading to cellular damage including those of the gastric mucosa that could lead to peptic ulceration (Burkeret al., 2003).Cooperative defence systems that protect the body from free radical damage include the anti-oxidant nutrients and enzymes. The antioxidant enzymes include catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx),and indirectly, glutathione reductase.Their roles as protective enzymes are well known and have been investigated extensively in both in vivo and in vitro model systems.The first three enzymes directly catalyse the transformation of peroxides and superoxides to non- toxic species.Glutathione reductase reduces oxidized glutathione to glutathione; a substrate for GPx.The consequences of oxidative stress are serious,and in many cases are manifested by increased activities of enzymes involved in oxygen detoxification (Kyounget al., 2003).Identification of new anti-oxidants remains a highly active research area, because recently reactive oxygen species generated in the cells of aerobically respiring organisms due to many factors have been implicated in the pathogenesis of many human sufferings like Parkinson’s disease, cancer and gastric diseases (Gutteridge, 1998; Mau et al.,2001; Gulcinet al.,2002b) and anti-oxidants may reduce the risk of various acute and chronic diseases caused by free radicals (Kyounget al.,2003). For the discovery of new leading agents which would be used for the benefit of mankind, we focused our study on the free radical scavenging potentials of Nigella sativa. The plant Nigella sativa has been used for medicinal purposes for centuries, both as a herb when pressed into oil in Asia, Middle East and Africa. It has been traditionally used for a variety of conditions and treatments related to respiratory health, stomach, intestinal, kidney, liver, circulatory and immune system support, and for general well-being. The seeds are used as carminative, aromatic, stimulant, diuretic, antihelminthic,galactagogue and to increase sweat. They are used as a condiment in curries. A tincture prepared from the seeds is useful in indigestion, loss of appetite, diarrhoea, dropsy, amenorrhoea, dysmenorrhoea and in the treatment of worms and skin eruptions. Externally, the oil is used as an antiseptic. To arrest vomiting, the seeds are roasted and given internally (Gupta et al., 2009).

2.0 MATERIALS AND METHODS:

2.1 Plant material:
Nigella sativadried seeds were obtained during the month of July, 2011 from Sabon-Gari market in Zaria. Botanical identification and authentification were done by Mr. U.A Gallah at the Herbarium section of the Department of Biological Sciences, Ahmadu Bello University, Zaria. A voucher herbarium specimen (No: 101201) was deposited at the herbarium for future references.

2.2 Extraction and fractionation of the plant material:
Nigella sativa seeds weighing about 2kg were crushed and pounded with pestle and mortar. The powder was extracted with aqueous ethanol (70%) in a Soxhlet Extractor, concentrated using rotaryevaporator at reduced pressure, suspended in ethanol and partitioned with ethyl acetate(ETAC) to obtain the ethyl acetate fraction. The fraction obtained was further concentrated in-vacuo and the residue obtained. The extract yielded about 80% of the residue fraction.

2.3 Phytochemical screening of the fraction:
The ethyl acetatefraction was analysed for the presence of
flavonoids, alkaloids, saponins, steroids, glycosides, anthraquinones, resins and reducing sugars using standard procedures for analysis (Evans, 2002 and Harborne, 2007).
2.4 Acute toxicity studies:
Lethal Dose (LD50) determination was conducted using the method of Lorke (1983). In the initial phase, male rats were divided into three groups of 3 rats each, making a total of 9 rats, for the ethyl acetate group.The rats were treated with the ethyl acetate fraction of the extract at doses of 10, 100 and 1000mg/kg subcutaneously. Animals were observed for 24 hours and the number of death(s) or those that showed neurological signs were recorded. In the second phase, the animals were grouped into 4 groups of one rat each and treated with the ethyl acetate fraction at appropriate doses subcutaneously. The rats were observed for 4 h for deaths or neurological signs, and the final LD50 was calculated as the square root of the highest non-lethal dose in which the animal survived multiplied by the lowest lethal dose in which the animal died.

2.5 Drugs and chemicals/reagents:
Cimetidine (LekPharma, Slovenia), Indomethacin (Liomethacin(R))(Cheisi, Egypt),
Thiopental Sodium (Abbott Laboratories,UK), Trichloroacetic Acid Solution 6% w/v (Sigma-Aldrich,USA),Phosphate Buffered Saline (PBS) Solution at pH 7.4 (Life Technologies,USA), Heparin (Pfizer Pharmaceuticals, USA), Potassium Phosphate at pH 7.5 (Sigma-Aldrich,USA), Thiobarbituric Acid 25mmol/L (Sigma-Aldrich,USA), Hydrogen Peroxide (H2O2) 500mM/L (OCI Chemical Corporation, USA), Ethylene DiamineTetraacetic Acid (EDTA) (DOW ChemicalCompany,USA), Trichloroacetic Acid Solution 6% w/v (Sigma-Aldrich,USA).

2.6 Experimental animals:
A total of fifty eightadult male albino wistar rats were used in this study. The animals were obtained from the Animal House, Faculty of Medicine, El-Kasr el-Ain, Cairo University, Egypt. Their weights ranged from 180 – 240g. They were maintainedunder a similar conditions of humidity, temperature and light/dark cycle respectively and each of the animal was kept in a single individual cage, with wide-meshed galvanized wire bottoms to decrease coprophagy as much as possible.The rats were given access to food and water ad libitum for two weeks to acclimatize, prior to the commencement of the experiment. The rats were treated in accordance to the Principles of Laboratory Animal Care, and experimental protocol was approved by the Animal Ethical Committee in accordance with the guide for the care and use of laboratory animals.At the time of the experiment, all treatments were conducted between 9:00 and 10:00 (GMT+1) h to minimize variations in animal response due to circadian rhythm. The animals were divided into the following groups and subgroups for gastric mucosal damage and gastric secretion studies respectively.

2.7 Experimentaldesign:
Group 1: Normal saline. Five rats received normal saline (1ml/kg/rat subcutaneously (S.C)).
Group 2: Indomethacin-treated.Five rats received indomethacin (20 mg/kg S.C)
Group 3: Cimetidine-treated. Ten rats for the study of the

for the study of the effect of cimetidine, 50 and 100 mg/kg S.C, given 30 mins prior to indomethacin administration (5 rats for each dose).
Group 5: Nigella sativa-treated. Fifteen rats for the study of the effect of ethyl acetate fraction, each at three different doses (50, 100 and 200 mg/kg S.C), when given 30 minutes prior to indomethacin administration (5 rats for each dose).

2.8 Collection of blood samples
Blood samples were collected using heparinized capillary tubes from the retro-orbital plexus of each rat, and kept in EDTA bottles. Immediately, the blood was centrifuged at 1,006 x g at 6oC for 10 minutes, the plasma was obtained and preserved at -20oC until use.

2.9 Collection of tissue samples
From each of the already dissected rats, the liver tissues were removed and placed in tissue sample bottles containing normal saline, ready for sample preparation as a homogenate for oxidative activity assay.

2.10 Determination of anti-oxidant enzyme activities.

2.11 Catalase assay: Determination of CAT activity was carried out according to the method described by Sinha (1972), Passatiet al., (1980) and Achi (1984), with Catalase Assay Kit (Bio-diagnosticR), Egypt. CAT. No. MD 25 17.Distilled water 0.9 ml was added to 0.1ml of tissue homogenate and mixed thoroughly. 2.5ml of phosphate buffer was put into a small conical flask; 0.5 ml of tissue homogenate was added; and 2.0ml of H2O2 also added, and a stop watch was started. The reaction mixture thoroughly mixed and the reaction stopped after every 60 seconds for 3 min with dichromate/acetic acid solution. The mixture in the flask was heated in a water bath for 10 minutes at 80oC. Absorbance was read at 570nm. Catalase activity was expressed as unit/mg of protein.

2.12 Superoxide dismutase assay
Determination of SOD activity was carried out according to the method described by Fridovich (1987), with Superoxide DismutaseAssay Kit (Bio-diagnosticR), Egypt. CAT. No. SD 25 21.To 0.1ml of tissue homogenate was added0.9 ml of distilled water to make 1:10 dilution of tissue homogenate. An aliquant mixture of 0.2 ml of the diluted microsome was added to 2.5ml of 0.05M carbonate buffer. The reaction started with the addition of 0.3ml of 0.3 mM adrenaline. The reference mixture contained 2.5ml of 0.05 M carbonate buffer, 0.3ml of 0.3mM adrenaline and 0.2 ml of distilled water. Absorbance was measured at intervals of 30 s up to 150 s at 480nm. SOD activity was expressed as unit/mg protein.

2.13 Determination of Lipid peroxidation (Malondialdehyde):
Lipid peroxidation was evaluated according to the procedure described by Ohkama and Ohishi (1979), as modified by Varshney and Kale (1996), with Lipid Peroxide Assay Kit (Bio-diagnosticR), Egypt. CAT. No. MD 25 29.To 1.0 g of stomach tissue, 10ml of 1/150 phosphate buffer (pH 7.0) was added and homogenized. 0.5ml tissue homogenate, 0.5ml saline and 1.0ml of 10% TCA was added.The mixture was centrifuged at 3000rpm for 20 min.0.25 ml of 0.1M TBA was added to the content and mixed well.The mixture was incubated for 1 h at 95oC and allowed to cool down and the supernatant 2.0 ml was measured using spectrophotometer at 532nm. The level of lipid peroxides was expressed as MDA nmol/mg of protein.

2.14 Statistical analysis
All data were expressed as Mean ± S.E.M (standard error of the mean) using SPSS Version 20. Statistical evaluation was done by analysis of variance (ANOVA) followed by post-hoc analysis by Duncan and Scheffe. Values of p<0.05 were considered significant (Microcal Software Inc., Northampton, USA).
RESULTS

Table 1: The Phytochemical Analysis of Ethyl acetate Fraction of Nigella sativa L. seed extract.

Phytochemical Tests Ethyl acetate
Test for flavonoids +++
Test for alkaloids ++
Test for tannins +
Test for saponins –
Test for steroids Trace
Test for glycosides –
Testforanthraquinones +
Test for reducing sugars +
Test for resins –

+ :Presence of the constituents – – Absence of the constituents

Effect of Ethyl acetateFraction on Markers of Oxidative Stress
As shown in table 2, administration of indomethacin 20mg/kg, the values obtained for catalase, superoxide dismutase and malondialdehyde were 4.76 ± 0.16u/mg, 6.21 ± 0.62u/mg and 4.53 ± 2.19nmol/mg of protein respectively. The catalase significantly increased compared to the normal saline control.The cimetidine 50mg/kg and 100mg/kg alone recorded 1.29±0.05u/mg and 5.98±0.30u/mg of catalase activity with the cimetidine 50mg/kg significantly (p<0.05) decreasing compared to indomethacin control, where as the cimetidine 100mg/kg significantly increased compared to the normal saline control. The superoxide dismutase and malondialdehyde in those groups are insignificant. Cimetidine 50mg/kg when administered with indomethacin 20mg/kg, catalase, superoxide dismutase and malondialdehyde values were 1.59 ± 0.13u/mg, 3.94 ± 0.11u/mg and 4.47 ± 2.52nmol/mg respectively, with the catalase significantly (p<0.05) decreasing, compared to the indomethacin control. In cimetidine 100mg/kg plus indomethacin catalase decreases significantly lower than the indomethacin control with a value of 4.15 ± 0.14u/mg, whereas superoxide dismutase and malondialdehyde were 2.75 ± 0.28u/mg and 3.816 ± 1.45 respectively, and both are insignificant.
In the ethyl acetate fraction 50, 100 and 200mg/kg plus the indomethacin 20mg/kg, catalase, superoxide dismutase and malondialdehyde values were 1.30 ± 0.15u/mg, 4.16 ± 0.51u/mg and 12.58 ± 1.82nmol/mg respectively for ethyl acetate 50mg/ug, while 1.83 ± 0.08u/mg, 3.56 ± 0.16u/mg and 14.30 ± 1.40nmol/mg were obtained for the ethyl acetate 100mg/kg respectively. For ethyl acetate 200mg/kg, 5.13 ± 0.47u/mg for catalase, 2.72 ± 0.28u/mg for superoxide dismutase and 4.50 ± 1.16nmol/mg for
malondialdehyde. The catalase level activity for ethyl acetate 200mg/kg was significantly higher than the control, ethyl acetate 50mg/kg and 100mg/kg groups.

DISCUSSION AND CONCLUSION
Recent clinical researches shed a light on the NSAID-induced mucosal injuries and the evident roles played by reactive oxygen species (ROS) via the free radical scavenging pro and antioxidant property of several plant species used in folkloric medicine.
Involvement of ROS in pathogenesis of gastric ulceration was first evident from the studies on ischaemiare-oxygenation-induced gastric mucosal injury (Yoshikawa et al., 1989; Yuda, 1993; Perry et al., 1996). The results of these experiments are in line with those previous reports. In this respect, the ETAC-treated groups, both 50 and 100mg/kg showed decreased catalase activity compared to control, decreased SOD activity that was dose-dependent and increased MDA concentration. Indomethacin-induced gastric ulceration was accompanied with a severe oxidative stress in gastric tissue causing damage to key biomolecules such as lipids, which was apparent from the stimulated lipid peroxidation that led to the accumulation of MDA as well as reduction in the gastric activity of CAT. As shown in the present results, the ETAC fraction of N.sativa significantly reversed the indomethacin-induced changes in SOD, CAT and MDA. This significant reduction in lipid peroxide levels along with significant increase in CAT level suggest increased antioxidant activity and prevention of lipid peroxidation by the ETAC fraction of Nigella sativa. Preventive anti-oxidant enzymes such as SOD and CAT are the first line of defence against reactive oxygen species (ROS) (Yogenderet al., 2007). Flavonoids and other phenolic compounds of plant origin have been reported as scavengers and inhibitors of lipid peroxidation (Formica and Regelson,1995) and are known to have the capacity to sequester endogenously and exogenously produced ROS and free radicals, thereby inhibiting mucosal damage (Repetto and Llesuy, 2002). This inhibition of lipid peroxidation and the generation of MDA and related substances from lipids that react with thiobarbituric acid was found to be inhibited by this extract. The inhibition was found to increase when the concentration of the extract was increased. Thus, it appears that the anti-oxidant property of this extract could counteract oxidative damage caused by NSAIDs. The cimetidine treated groups were insignificant.
It has been shown that compounds isolated from Nigella sativa have appreciable free scavenging properties (Buris and Bucar, 2000). These might be attributed to various mechanisms among which are prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxide, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging ability (Diplock, 1997; O’Ktayet al., 2003). The ethyl acetate fraction exhibited free-radical scavenger activity as well as primary antioxidant that react with free radicals, which might possibly limit free radical damage, occurring in the body. ETAC fraction of Artemesiaapiacea was found to possess strong antioxidant activity and the major metabolite isolated was flavonoid cacticin (Lee et al., 2002). Similarly, the extracts from Culcitumreflexumhave been reported to possess photo-protective and anti-oxidant activities and their major compounds were flavonoids (Aquino et al., 2002). In line with the present findings, many researchers have indeed demonstrated the in vivo and in vitro anti-oxidant and free radical scavenging activities of flavonoids (Cao et al., 1997; Rice-Evans et al., 1997; Brown et al., 1998; Plumb et al., 1999; Pietta, 2000). Matsuda et al. (2003) reported that flavonoids are the major secondary metabolites class with several descriptions of antiulcer, antioxidant and other gastroprotective properties. Essential oils which are mixtures of wide variety of volatile terpene hydrocarbons (aliphatic and cyclic) were reported in N.sativa(Magiatiset al.,2002). Several terpenoid-containing extracts from plants commonly used in non-orthodox medicine to treat different gastric illnesses have been reported (Arrietaet al., 2003; Schmerda-Hirschmannet al.,2005; Morikawaet al.,2006). According to Esteveset al. (2005), the essential oil from Cascariasylvestris, which contains sesquiterpinebicy-clogermacrene as the major component, displays mucosal cytoprotectiveproperty. Baccharisdra-cunculifolia essential oil also showed a similar activity (Klopellet al., 2007; Lemosetal., 2007).

CONCLUSION
These studies suggested that the ETAC fraction of Nigella sativa protects against free radicals and possessed appreciable free radical scavenging property by promoting oxidant/antioxidant balance,hence it might play a role in combating gastric ailments like peptic ulcers.

R.M. Gali, F. Dalhatu, Y.P. Mamza, F. Zakari
Department of Medical Laboratory Science, College of Medical Sciences, University of Maiduguri, Borno State Nigeria.
S. Kwari
Department of Obstetrics and Gynaecology, College of Medical Sciences, University of Maiduguri, Borno State.
R.Y. Genesis
Department of Chemical Pathology, University of Maiduguri Teaching Hospital Borno State Nigeria.

All correspondents to: Dr (Mrs) R M Gali, Department of Medical Laboratory Science, College of Medical Sciences,
University of Maiduguri. E-Mail rmgali@yahoo.com

ABSTRACT
Objectives: This study assessed the effect of contraceptives on glucose and lipid profile of women on contraceptives (oral and injectable) in family planning unit of State Specialist Hospital, Maiduguri, Borno State Nigeria.
Methods: Ninety women made up of Fifty(50) women) on oral and injectable contraceptives and forty (40) mothers who are not on any contraceptive had their Glucose determined by glucose oxidase method, total cholesterol, triglyceride and high density lipoprotein determined by enzymatic reaction while Freidwald’s formular was used for low density lipoprotein estimation.
Results: Fasting glucose level was significantly higher in contraceptive women than in non-contraceptive women (5.54±3.35mmol/L and 4.15±0.70mmol/L respectively; p=0.018). Total cholesterol was significantly higher in contraceptive women than in non-contraceptive women (5.33±1.31mmol/l and 4.60±1.12mmol/l; p=0.007). In addition, low-density lipoprotein were significantly higher in contraceptive women than in non-contraceptive women (3.43±1.22mmol/l and 2.65±1.14mmol/l; p=0.007). Whereas triglyceride and high-density lipoprotein in both group were statistically not significant (p>0.05). The comparison of oral and injectable contraceptives showed no significant differences in all the parameters studied except high density lipoprotein which is higher in oral contraceptive women than in women using injectable contraceptives (1.70±0.73mmol/l and 1.37±0.37mmol/l; p=0.043). There was no significant differences in Glucose concentration of women on oral contraceptives and those on injectable contraceptives (P>0.05),
Conclusion: In conclusion, glucose, total cholesterol and low-density lipoprotein are elevated in this study. Therefore, regular tests for lipid profile and glucose is necessary for women on contraceptives to avoid risk of developing diabetes mellitus and cardiac vascular disorders.

KEYWORDS: Glucose, lipid profile, contraceptives.

INTRODUCTION

Birth control in developing countries has decreased the number of maternal deaths by 40% and could be prevented by 70% if fully demands for birth controls were met1,2. Birth control methods include barrier methods, hormonal birth controls, sterilization, and behavioral methods. Hormonal contraceptives are referred to as birth control methods that act on the endocrine gland. Hormonal contraceptives work by inhibiting ovulation and fertilization3. Hormonal contraceptives are available in a number of different forms. The oral and injectable methods are by far the most popular methods. Altogether, 18% of the world`s contraceptive users rely on hormonal method4. The use of injectable hormonal contraceptives has risen rapidly
in many countries5. The estimated number of injectable users doubled from 1995 to 2005; by 2015, about 40 million women are expected to use this method5. Some studies indicated that intake of oral contraceptives could cause metabolic changes in carbohydrate, protein, and lipid macromolecules6,7. Progestin exerts an adverse influence on lipid metabolism, they have ability to counteract the estrogen-induced changes in low density lipoprotein and high density lipoprotein levels8. Women are prone to develop dyslipidemia due to long-term use of hormonal contraceptives9. Hormonal contraceptives induce the body to produce more of the stress hormone cortisol (glucocorticoid), which has effects on lipid and glucose metabolism. Therefore, this study aims at assessing the effect of contraceptives on glucose and lipid in women in this environment.

MATERIALS AND METHODS
This study was carried out in State specialist hospital (family planning unit) Maiduguri Borno State Nigeria. A total of 90 women were recruited for this study. Fifty women who were randomly selected at the family planning unit and forty women who were not on any contraceptives were regarded as control group. The subjects were apparently healthy and were within the reproductive age. Eleven (11) of them were on combined oral contraceptives, whereas 39 were on injectable contraceptive (Medroxyprogesterone). Ethical clearance was obtained from the research and ethical committee of State Specialist Hospital Maiduguri. Oral consent of the mothers was sought after explaining the purpose of the research. Questionnaire was administered to every woman that was eligible for the research.
A fasting blood sample of 5ml volume was withdrawn from each patient; 2ml of blood was collected into fluoride oxalate vacutainer bottle and 3ml into plain container. The plasma was used for the estimation of fasting glucose using oxidase-peroxidase enzymatic colorimetric method as described by Trinder10 and the serum was used for the estimation of triglyceride (TG) by enzymatic method as described by Fossati11, total cholesterol by enzymatic method as described by Meiattini12 and HDL by enzymatic method as described by Warnick13. All the test kits are manufactured by Randox laboratories limited, Ardmore, Diamond road, Crumin.co. Antrim UK. Low-density
lipoprotein (LDL) was calculated using Freidwald’s formular as described by Freidwald14. Statistical analysis of data was done with SPSS version 18 to determine mean, standard deviation, Anova and student t- test. The level of confidence was set at p<0.05.

RESULTS
There were significant differences between means of fasting plasma glucose, total cholesterol, and low-density lipoprotein of contraceptive women with that of the control group. Fasting plasma glucose level was significantly higher in contraceptive women than non-contraceptive women (5.54±3.35mmol.l and 4.15±0.70mmol/l; p=0.018).There is significant high level of total cholesterol in women on contraceptives than non contraceptive (5.33±1.31mmol/l and 4.60±1.12mmol/l; p=0.007). Also low-density lipoprotein is significantly higher in women on contraceptive than the women that are not on contraceptive (3.43±1.22mmol/l and 2.65±1.14mmol/l; p=0.007). On the other hand, there was no significant differences in the levels of triglyceride and high-density lipoprotein in both women on contraceptive and the control group. The comparison between women on oral contraceptive and those on injectable contraceptives showed no significant difference in all the parameters study except high-density lipoprotein, which is significantly higher in women on oral contraceptives than those on injectable contraceptives. No significant change was seen in all the parameters on duration of the use of contraceptives.

were slightly higher in oral contraceptive users than in injectable contraceptive women but are statistically non significant. However, high-density lipoprotein cholesterol was significantly increased in oral contraceptive women as compared to those injectable contraceptive women (Table 2), and this may be due to the effect of estrogen in combined oral contraceptive pills. This study is in accord with the findings of Sitruk-ware19 and Hogan et al20 whose studies revealed high levels of high-density lipoprotein cholesterol in oral contraceptive women.
This study grouped the subjects for the duration of the use of contraceptives into two; less than five years using contraceptive (45 subjects) and five to ten years of using contraceptives (5 subjects). Our findings revealed no significant changes in all the parameters used on duration of contraceptives use (Table 3), this study contradicts the findings of Berenson et al18 who stated that over 3 years of using injectable contraceptives, there was increased in levels of glucose and insulin. In addition, our results contradict with other studies who stated that the levels of triglyceride, LDL-cholesterol and VLDL-cholesterol increased with duration of oral contraceptives intake 16-18. Although this study revealed increased in low-density cholesterol due to duration, the increase is not statistically significant. On the other hand, there is decreased in high density cholesterol in 5-10 years of contraceptives use (Table 3) but not statistically significant. This study was not able to prove the claim by Santos et al9 who state that Women are prone to develop dyslipidemia due to long-term use of hormonal contraceptives.
In conclusion, contraceptives are good for birth control that help families to plan their lives, this should be used with caution, and regular check up is necessary to avoid risk of diseases such as diabetes mellitus and cardiovascular diseases that may be detrimental to human health.

ACKNOWLEGDMENTS
We wish to thank all staff of Family planning unit of State Specialist Hospital Borno State and Department of Chemical Pathology University of Maiduguri Teaching Hospital Borno State Nigeria for their support and
DISCUSSION
The major health risks of oral and injectable contraceptives are glucose impairment and cardiovascular diseases particularly coronary artery disease, stroke and venous thrombolism. Hypercholesterolemia is associated with endothelial cell dysfunction, elevated oxidant stress, and creation of a strongly pro-inflammatory conditions15.
In this study, the total cholesterol and low-density lipoprotein cholesterol levels in women on contraceptive are higher than non-contraceptive women and are statistically significant (Table1). This study agrees with the findings of Yesmin and Asare16,17 who reported increase total cholesterol, and low density lipoprotein. The increase in serum total cholesterol in women taking contraceptives may be due to impaired lipoprotein metabolism and higher levels of low-density lipoprotein may be due to increase lipoprotein synthesis rather than impaired lipolytic catabolism in association with accumulation of cholesterol16. In this study, there is no significant difference in triglyceride level in women on contraceptive and non contraceptive women (Table1), which is in line with findings of Asare17 and contradicts the findings of Yesmin16, who stated that there is elevated triglyceride in women on contraceptive than non contraceptive women. He attributed that increased serum triglyceride might be due to increase production and transportation of very low-density lipoproteins that endogenously synthesize triglyceride in the blood; although our finding did not indicate that.
There was no difference in high-density lipoprotein, which is in accordance with findings of Asare17. In this study, fasting plasma glucose was significantly high in contraceptive women than non contraceptive women, this finding is similar to those of Berenson et al18 who reported high glucose level in women on contraceptives. They attributed this high glucose level to compensation for increased insulin resistance and glucocorticoid-like activity of progestogen.
Glucose level in oral contraceptive women was slightly higher than in injectable contraceptive women but is statistically non-significant. Also the total cholesterol, triglyceride, and Low Density Lipoprotein cholesterol

Kudi A.A., Ahmed R.A., Baba-Ali F.

Bio-global Diagnostic & Research Laboratories, Opp. State Specialist Hospital Main Gate, Jekadafari Road, Gombe
Uba A., Tahir F., Yusuf I.Z.

Department of Medical Microbiology & Immunology Federal Teaching Hospital Gombe, Gombe State
Bubalu J.L

Faculty of Science, Biological Science Department Abubakar Tafawa Balewa University, Bauchi

All correspondence to: Kudi A.A, kclifeglobal@yahoo.co.uk

ABSTRACT
Background
Brucellosis is a disease of domestic, livestock and wild animals with serious zoonotic implications in man. Its spread is worldwide and transmission to humans is by contact with fluids from infected animals or derived food products such as unpasteurized milk and cheese. The clinical picture of the disease in man is so strange and protean; easily bewildered with other infectious and noninfectious diseases, leading to diagnostic delays and late onset of therapy. The intention of this study was to determine and document the prevalence of Brucellosis in patients presented with acute febrile illnesses resembling malaria and/or typhoid infections using the serological technique.
Methods: A retrospective study of a two-year laboratory records of cases of brucellosis among patients with febrile illnesses resembling malaria and/or typhoid fever seen at the General out-patient department (GOPD), Federal Teaching Hospital, Gombe between 2012 and 2014. Blood samples routinely collected from 246 patients were allowed to clot and the sera extracted were tested for Brucella antibodies using Rose Bengal plate test (RBPT). Standard test protocol described by Alton et al., (1975) and Morgan et al., (1978) was followed. Results obtained were statistically analysed using tables and chi-square test at 95% confidence level.
Results: Out of the 246 samples analysed, 56.6% were from the males, while 43.4% from females between the ages 0-60yrs. Our record shows 32.5% positivity for brucellosis, while the remaining 67.5% were negative. Among the males tested, 14.5% were found positive for the disease, while among the females 18.0% was recorded, (p>0.05). Majority (13.3%) that tested positive for brucellosis were between the ages 31- 40 years. The least (3.6%) affected were seen in ages 21-30 and 51-60 years.
Conclusion: Brucellosis among the population tested was considered to be high. Symptoms clinically thought to be malaria and/or typhoid fever were seen to be cases of brucellosis. There was no significant association between sex and age in the rate of infection (p>0.05). Routine laboratory test request for Brucella antibody among patients with febrile illness in our Community is recommended. Co-ordinated public enlightment on zoonosis, with special emphasis to brucellosis in Gombe State was encouraged.

Key words: Brucellosis, Zoonosis, Febrile illnesses, Pyrexia of unknown origin, Gombe

INTRODUCTION

Brucellosis is an infectious disease caused by bacteria of the genus Brucella (Colmenero et al., 1990; Memish et al., 2000; Al Dahouk et al., 2003; Kaltungo et al., 2014). The disease is caused by several species of Gram-negative facultative intracellular bacteria of the genus (Sofian et al., 2008). It is a neglected debilitating zoonosis and is recognized as an occupational hazard with a high prevalence in developing countries
(Mabel et al., 2013). Primarily, it is a disease of domestic, livestock and wild animals with serious zoonotic implications in man; causing huge economic losses to the livestock industry. Cattle, goats, pigs, sheep, horses and dogs play an important role in the transmission of this disease to man. It is defined as a contagious systemic bacterial disease of ruminants, characterized by inflammation of the genital organs and fetal membranes, abortion, sterility and formation of localized lesions in the lymphatic system and joints (WHO, 1971, CDC, 2005).

Brucellosis remains a main source of disease in humans and animal husbandry worldwide (Corbel, 1997).

The clinical picture of brucellosis in man is so strange and protean that it can be easily bewildered with other infectious and noninfectious diseases, leading to diagnostic delays and late onset of therapy (Al Dahouk and Nockler, 2011). Malta, Rock, Gilbraltar, Crimean, Cyprus or Mediterranean fever, Bang’s disease, intermittent typhoid or Typho-malarial fever, undulant fever, etc, are just various synonyms for brucellosis (Al- Dahouk et al, 2003). Those suffering from the disease show unspecific symptoms, e.g. fever, chills, malaise, arthralgia, headache, tiredness and weakness. Therapeutic failure and relapses, chronic courses and severe complications like bone and joint involvement, neurobrucellosis and endocarditis are characteristic for the disease (Al- Dahouk et al, 2003).
About half a million human brucellosis cases are reported annually. However, according to the WHO (1997) estimates, the true frequency of the disease is 10-25-times higher than the reported number. The highest annual incidence rates are reported from the Middle Eastern countries, such as Syria, Iraq, Iran, and Saudi Arabia (Pappas et al., 2006). In Iran, where Brucellosis is endemic, the incidence of the disease is up to 34 per 100,000 per year in certain areas (Najafi et al., 2011). In Nigeria, it was reported that human brucellosis is hardly diagnosed in hospitals despite suggestions that the magnitude of the infection may be greater than appreciated (Njoku 1995; Rajis et. al, 2003).
Detailed studies confirming the problem of brucellosis in Nigeria’s livestock have been documented by several authors (Esuruoso, 1974; Falade, 1974; Falade, et al., 1974; Falade et al., 1975; Okon, 1980, Chukwu, 1987; Brisibe et al., 1993; Ajogi, 1997; Ajogi et al., 1998; Ogundipe et al., 1994; Ishola et al., 2001); with evidence of the spread of the disease in all parts of the country which is usually accompanied by severe economic losses. Serological prevalence rate of between 0.20% and 79.70% have been reported in various parts of the country to date. The infection has been reported in various animal species in Nigeria (Esuruoso and Hill, 1971; Esuruoso, 1974; Falade, 1974; Falade and Shonekan 1981; Falade et al., 1975; Okoh et al., 1978; Adamu and Ajogi, 1995). These demonstrate how brucellosis has been identified as an endemic and problematic disease in Nigeria. However, the infection is not static; it is evident from previous studies that prevalence varies at different times and locations. This is especially apparent where there is no control policy, like Nigeria. There is a pattern of low and high prevalence in specific areas of the country and prevalence variability also arises between herds in the same area (Nuru and Dennis, 1975). Although prevalence in brucellosis has been shown to be low in most dairy and private farms, it is actually on the increase among nomadic and semi-nomadic herds which contribute about 95% of all annual food population in Nigeria (Rikin, 1988).

Making a diagnosis of brucellosis may be difficult because of the unspecific symptoms and signs shared with other febrile illnesses, slow growth rate of the causative agent in blood culture, and the complexity of sero-diagnosis (Colmenero et al., 1990; Memish et al., 2000; Al Dahouk et al., 2003). Presumptive diagnosis of brucellosis can be
made by the use of several serological tests to brucella antibodies, but the “gold standard” remains isolation and identification of the bacterium. Evidence of the infection either through serological or cultural examinations has been demonstrated in domestic livestock and humans in Nigeria (Ocholi et al., 1993). Most of the disease reports originated from Government herds where screening tests were easily carried out, while some originated from settled Fulani herds and private farms (Ocholi et al., 1993). The general situation is that the disease is more prevalent in Government-owned farms than in the nomadic herds (Esuruoso, 1974). Epizootiological investigations also revealed that results obtained varied depending on the region, area or animal group sampled (Ocholi et al., 1993). The distribution of the disease among humans is not well known but serological evidence has shown that the disease exists worldwide. Evidence of the presence of the disease in humans in Nigeria has also been published (Collard, 1962, Alausa, 1977, Alausa and Osoba, 1977, Falade, 1974 and Falade, 2002). It is a zoonosis and the disease in man is highly debilitating, though not considered to be fatal (Falade, 1974). Collard (1962) documented the first case of human brucellosis in Nigeria where Brucella antibodies were demonstrated in the sera of healthy persons in various parts of the country.
The intention of this study was to determine and document the prevalence of Brucellosis in patients presented with signs and symptoms of being ill (acute febrile illness) resembling malaria and/or typhoid infections in our hospital using the serological technique retrospectively. We suggest that similarities in signs and symptoms of diseases have often led to under diagnosis or misdiagnosis of brucellosis in humans. Most often, as the case may be, patient illness is dismissed as malaria, typhoid or pyrexia of an unknown origin (PUO); in that case, the disease go undiagnosed and untreated, leading to considerable suffering for those affected (John et al, 2002). We view brucellosis as one of the neglected zoonosis and most under reported or misdiagnosed disease in most hospitals in our locality. We found no known documented report of brucellosis among humans in Gombe and its environs; the few found reports were among domestic animals but not humans. Access to current documented cases of brucellosis in humans in our environment may be of paramount assistance in patients’ management thereby, contributing to quality healthcare delivery; the gap we made attempt to close.

MATERIALS AND METHODS
Study Area
Gombe, the capital city of Gombe State is located in the Guinea Savannah of North-Eastern part of Nigeria in West Africa. The State is bordered by Borno State to the East, Yobe State to the North-East, Taraba State to the South, Adamawa State to the South-East and Bauchi State to the West. It occupies a total land area of 20,265 square kilometres and has a population of about 2,353,879. The State is made up of 11 Local Government Areas (Jibrin, 2003, GomSACA, 2008). The principal ethnic groups of State are the Tera, Tangale, Fulani, Waja and Bolewa. The people are predominantly farmers and as is the culture, they keep and rear domestic animals and poultry for economic purposes.

Ethical Consideration and Confidentiality
Approval for this work was obtained from the Federal Teaching Hospital (FTH), Gombe Research and Ethics Committee. Patients’ consent was obtained at the time of sample collection to run the test. Throughout the test analysis, confidentiality of health information of the patient was maintained.

Sample Collection
Whole blood samples were collected from 246 patients with febrile illnesses resembling malaria and/or typhoid seen at the General out-patient department (GOPD), Federal Teaching Hospital, Gombe from 2012 to 2014. The samples were routinely collected in the Medical Microbiology & Immunology Laboratory of the same hospital by the attending Phlebotomist. From each patient, 4mls of whole blood was collected in a sterile screw capped non-anticoagulant 5ml-capacity disposable (plastic) bottles using sterile disposable syringe with a 21G needle. The blood was allowed to clot and then centrifuged at 1500 rpm to separate the serum from the red blood cells. Using a sterile disposable Pasteur pipette, the serum extracted was transferred into a sterile screw capped vial, labeled with a laboratory number that correspond with the subject identification. The labeled serum sample was ready for analysis (Alton et al., 1975). Where there was delay in the analysis, the sample was stored at -20oC until required for testing. Demographic characteristics of each patient were noted at the time of sample collection.

Serological Testing
Each sample was screened for Brucella antibodies using Rose Bengal Plate Test (RBPT). The test was carried out using the standard protocol described by Alton et al., (1975) and Morgan et al., (1978). Procedure: Serum samples and antigen were brought to room temperature (22 ± 4°C). Approximately 25µl of each serum was placed on a white tile and an equal volume of antigen was placed near each serum spot. Both were mixed thoroughly using a clean wooden rod and read for agglutination immediately after a 4-minute period. The agglutination reactions were recorded as positive (+) or negative (-) depending on whether there was agglutinations or not. The reagents in the kit were reconstituted and the test procedure was carried out according to manufacturers’ instructions. Individuals were considered as positive based on a positive RBPT result. The necessary quality control (QC) was carried out on the reagent used using known positive and negative controls. All safety precautions were followed according to the manufacturer’s instructions on the kit insert.

Statistical Analysis
Data collected were analysed using tables and chi-square tests. Results were considered as significant if the chi-square p-value was < 0.05 otherwise, non-significant if the p-value was > 0.05.

DISCUSSION
Similarities in signs and symptoms of diseases have often led to under diagnosis or misdiagnosis of brucellosis in humans. It was postulated that making a diagnosis of brucellosis is difficult because of the unspecific symptoms and signs shared with other febrile illnesses (Colmenero et al., 1990; Memish et al., 2000; Al Dahouk et al., 2003). The clinical picture of brucellosis is so strange and protean that it can be easily bewildered with other infectious and noninfectious diseases (Al Dahouk and Nockler, 2011). As the case may be, patient illness is dismissed as malaria, typhoid or pyrexia of unknown origin (PUO). This leads to diagnostic delays and late onset of therapy; therefore, laboratory investigation is always needed to confirm etiologic agent (Al Dahouk and Nockler, 2011).

Our findings in this study show that more males (56.6%) presented with febrile illness that resembles malaria and/or typhoid fever at our Hospital’s out-patient department (GOPD) than females (43.4%). However, there was no significant difference between the sexes in the clinical presentation (p>0.05). As high as 32.5% prevalence rate of brucellosis in this study was recorded among the patients tested. This finding seems to agree with other workers who reported – though in domestic animals, serological prevalence rate of brucellosis of between 0.20% and 79.70% in various parts of Nigeria (Esuruoso and Hill, 1971; Esuruoso, 1974; Falade, 1974; Falade and Shonekan 1981; Falade et al., 1975; Okoh et al., 1978; Adamu and Ajogi, 1995). Other researchers recorded in humans close to the range of 28 to 57% among high risk group such as abattoir/butchers workers (Falde 1974; Ocholi 1993; Edu, 2005). In contrast however to our findings, Maryceline et al., (2001) recorded as low as 5.2% sero-positivity among
patients with PUO in Maiduguri, northeastern Nigeria. Perhaps geographical location and other demographic factors play some roles to this variation. The distribution of the disease among humans is not well known but serological evidence has shown that the disease exists worldwide and it is a zoonosis (Falade, 1974). Our findings therefore, has further agrees with other published reports that the presence of the disease in humans in Nigeria exist (Collard, 1962; Alausa, 1977; Alausa and Osoba, 1977; Falade, 1974; Falade, 2002). The first case in Nigeria was documented by Collard (1962) among apparently healthy persons in various parts of the country.
Other workers further reported cases among abattoir workers, nomads and those handling meat and other animal products. Mabel et al. (2013) recorded 32.7% seroprevalence among butchers in Abuja Nigeria; this is almost comparable to our findings in this study. Asanda and Agbede (2001) suggested that infection seems more associated with humans engaged in livestock and livestock product activities than those engaged in other productive ventures. Occupational status of the subject in this study was not determined; we therefore acknowledged this as one of the limitations, however, we could assume that contact with infected animals or animal products, such as meat, milk and other animal products among the population was inevitable, hence the high prevalence recorded. It was reported that the infection is not static; the prevalence varies at different times and locations (Rikin, 1988).
We noticed in this study that the females have the highest prevalence of brucellosis with 18.0% as against the males (14.5%). However, there was no significant difference in the rate of infection among the sexes (p>0.05). Our findings here tally with the report of Maryceline et al., (2001) where sex has no significance role in the distribution of brucellosis. This is in contrast with the findings recorded by Marbel (2013) among the abattoir workers and butchers in Abuja Nigeria, where males were significantly more infected with Brucella. This should be expected since males (in terms of occupation) are more involved in the handling of animals and animal products such as raw meat, unpasteurized milk and cheese. Such activities have been documented as significantly associated with Brucella infectivity (Hannah et. al., 2011); butchering in particular was noted to be a male-dominated activity thereby, the males stand at high risk of being infected with Brucella.
Our findings indicated that the highest seropositivity (13.3%) was in the age group 31-40 years, while the least was found in the ages between 21-30 and 51-60 years. However, in contrast to our findings, other workers reported individuals who tested positive to the brucellosis test were within the ages 18-25 years, while the least affected were those above 41 years of age (Marbel, 2013). The differences in the rate of infection among the ages in this study was found to be statistically significant (p<0.05). Reasons for the difference in the rate of infection was not immediately known to us, but it could be speculated that the ages 31-40 years is the most productive and active years thereby, frequency in contact with infected animals/products was high, as such, chances of getting infected therefore, most likely was significantly increased. Maryceline et al., (2001) however reported that age does not significantly affect the distribution of brucellosis.he limitation to the current study as observed and stated earlier was lack of determining the level of association of the subject with animals/animal product and brucellosis to confirmed zoonosis. However, infection with Brucella has always been reported to be the primary risk factor associated with keeping sheep, goats and other small animals in the household (Hannah et. al., 2011). It was generally observed that keeping and rearing goats, sheep, pigs and Cattles in our community is the practice for economic reasons; we may therefore, speculate that the high seropositivity (32.5%) recorded in our study was somewhat expected. The study was also limited in determining the level of knowledge of brucellosis in the community. Howbeit, with the high prevalence rate of infection recorded in this study, it is most likely that the level of awareness among the subjects, especially the zoonotic role in transmission, control and preventive measures was very low. In such situation therefore, and with the fact that infection is often subclinical, with high morbidity for both humans and animals (Colmenero et al. 1996), we again speculate that the disease is wide spread and diagnosis is either missed, under diagnosed or unreported in most of our healthcare settings.
Conclusion
The study reveals that among the population tested for brucellosis, there was no significant association between sex and age in the rate of infection. To the contrary however, other workers have reported significant difference in the rate of infection among the male abattoir workers/butchers (Falde 1974; Ocholi 1993; Edu, 2005). Among all that presented with febrile illnesses in our Centre, 32.5% would have been thought, dismissed or treated as having malaria, typhoid fever or PUO, but for the serological test which indicated brucellosis. This shows the significance of Brucella antibody testing on all patients presented with acute febrile illnesses in our healthcare facilities. We therefore advocate regular or routine laboratory test requests for diagnosis of brucellosis as is the case with other endemic infectious diseases, such as malaria and typhoid fever in our Community. The practice may enhance adequate and prompt diagnosis of acute febrile illnesses. Co-ordinated public enlightment and education programme generally on zoonosis, with special emphasis to brucellosis’ prevention and control should be instituted by the authorities concern in Gombe State and North eastern Nigeria as a whole.

Jemikalajah J.D.
Department Of Medical Microbiology And Parasitology, Faculty of Clinical Science, Delta State University, Abraka.
Adu M.E.
Department Of Medical Laboratory Services, Antiretroviral Therapy Centre, Central Hospital, Agbor.

All Corresponding to: adumatthew10@yahoo.com

ABSTRACT

Pregnancy induced changes in the maternal system in order to accommodate the foetus. One of such changes is the anabolism and catabolism of plasma proteins. The effects of pregnancy on serum proteins were investigated in eighty (80) pregnant women attending antenatal clinic in central hospital, Auchi and forty (40) apparently healthy women as controls. Total protein, albumin, and globulin as well as albumin/globulin ratio were examined. There was decreased total protein, albumin and albumin/globulin ratio and increased globulin in pregnant women when compared with non- pregnant women. Also there was significant difference when different trimesters were compared. These changes are attributed to increased plasma volume as well as synthesis of pregnancy induced plasma proteins. We advocate the routine examination of serum protein during pregnancy.

Keywords: Serum proteins, Pregnancy, Globulin, Auchi, Trimester

INTRODUCTION

Pregnancy involves the sequence of events that take place after fertilization of an ovum, thus enabling the fertilized ovum to eventually develop into a full- term foetus (Guyton and Hall, 2000). During pregnancy estrogen and progesterone are produced in large quantities and exert some influence on a pregnant woman. These hormones induce an increase in the metabolism of the woman as she prepares to carry the baby (Bassi et al., 2011). Physiological response in the metabolism of the expectant mother is due to foetal requirement of oxygen and food substance, growth of the uterus and preparation for lactation. The pregnant women experience physiological changes to support fetal growth and development (Blackburn and Loper, 1992, Taylor 1995). The level of estrogens (estradiol) and progesterone increase progressively during pregnancy (Vanthiel and Gavaler, 1987). These sex hormones have effects on hepatic metabolic, synthesis, and excretory functions (Vigil and Gratia, 2004, Bacq, 1999, Marpeau et al., 1999). The phenomenon of hemodilution secondary to the increase in plasma volume decreases the serum protein concentrations. Consequently, certain changes in values of liver function tests occur during normal pregnancy (Everson 1998, Alonso 2006). There are scanty literatures on the serum proteins of pregnant women especially in the locality studied; we therefore sought to determine the serum proteins of pregnant women in Auchi, Edo State, Nigeria.
MATERIALS AND METHODS
Study Area
This study was carried out in Central Hospital, Auchi, Edo State Nigeria. It is a secondary health institution that serves as a referral centre for other primary health institution in this locality.
Sample Collection
A total of 120 samples were collected from 80 pregnant women attending ante-natal clinic and 40 non pregnant women as controls after informed consent. Five milliliters of venous blood was collected aseptically into a plain container and allowed to clot. This was spun at 3000rpm for 10minutes to obtain a clear serum which is kept frozen until required for analysis. Ethical approval was obtained from the ethics committee of Central Hospital, Auchi.
Biochemical Analysis
Total serum protein was determined spectropho-tometrically using Biuret method (Doumas et al., 1981) while serum albumin was determined by bromocresol green method (Doumas et al., 1981). Serum globulin was calculated by subtracting albumin from total protein. The Albumin Globulin ratio was also determined by dividing Albumin with Globulin. All reagents were products of Randox Laboratories UK. In all test, manufacturer instructions were strictly adhered to.
Statistical Analysis
The groups mean ± SD was calculated for each analyte and significant difference between means evaluated

using the student t-test. Statistical Package for Social Science SPSS version 16.0 software (SPSS Inc., Chicago, IL USA) for windows was used, with P<0.05 considered as statistically significant.

RESULTS
The mean serum total protein of pregnant women was observed to be decrease (6.23±0.69) than the control subjects (6.63±0.43). Mean serum albumin level of pregnant women was found to be 3.43±0.61as against controls which is 3.93±0.36.Table 1 also show the globulin level of both pregnant women and controls to be 2.75±0.29 and 2.70±0.24 respectively. There was decreased Albumin: Globulin ratio observed in pregnant women (1.27±0.29) when compared with control subjects (1.46±1.50).
A/G = Albumin: Globulin Ratio
Fig 1 shows the mean serum proteins of different trimesters with first trimester having an increased total protein when compared with both second and third trimester. There is an increased serum albumin: globulin ratio observed in the third trimester when compared
with first and second trimester.

DISCUSSION
Pregnancy is a period between conception and delivery and has been associated with increased dietary protein requirements in humans. During this period of rapid growth, the foetus and placenta accrue proteins very rapidly (Lewis et al., 2010). Pregnancy induces major physiological, hormonal and biochemical changes to achieve an optimal outcome for the baby and its mother. Our study show significant decreased in total protein of pregnant women when compared with controls. This decrease can be attributed to changes in plasma volume as opined by Varley et al., (1980). The fall in protein concentration seen during pregnancy may be due to the dilution of the plasma, since total protein concentration is inversely related to plasma water concentration (Paaby 1960).

The albumin level of pregnant women was found to be significantly decreased when compared with non-pregnant women. This is in accordance with the findings of Coryell et al., (1950) and Horme et at., (1970) who attributed the decrease in albumin to increased plasma volume found in pregnancy. However, Laurell et al., (1968) stated that albumin synthesis which is normally stimulated due to hypoalbuminemia is inhibited during pregnancy by progesterone and estrogen which is abundant in pregnant women.

There was no significant difference observed in the globulin levels of pregnant and non pregnant women. This is in consonance with the findings of Adedeji et al., (2012) who observed that concentration of serum globulin is not affected by pregnancy. The Albumin/Globulin ratio of pregnant women was significantly decreased when compared with non pregnant women. This similar observation was reported by Adedeji et al., (2012). This difference is attributed to the decreased albumin as occasioned by increased plasma volume.
Our results revealed that the mean concentrations of serum proteins exhibited some variations with advancing gestational age. There was significant increased observed in the total protein of first trimester when compared with second and third trimester. Also there was significant increase in the total protein of second trimester when compared with third trimester. This is in tandem with Adedeji et al., (2012) who did similar work on protein profile of pregnancy. Shimokawa and his colleagueset al et al.,(1980) demonstrated that certain serum protein fraction of molecular weight of 185,000 Dalton are elevated in relative concentration during the first trimester and decrease to non- pregnant levels after 25th week of gestation. There was no significant difference observed between the first trimester and second trimester in albumin level when compared but there exist a significant difference when both first and second trimester where compared with third trimester. This is in agreement with previous findings of Adedeji et al., (2012).This difference is attributed to increased plasma volume being experienced in the third trimester as well as nutritional needs of the foetus. The globulin fraction of first trimester and second trimester was observed not to be significantly different but an increased significant difference was observed when third trimester was compared with both first and second trimester. This difference may be due to the synthesis of various pregnancy induced proteins such as ?- fetoprotein, placental isoenzyme of alkaline phosphatase, oxytocinase, human chorionic gonadotropin and the pregnancy associated plasma proteins. The concentrations of these proteins rise during gestation and disappear within few days or weeks of postpartum as opined by Joseph et al., (1978). The albumin: globulin ratio of the third trimester was significantly increased when compared with both first and second trimester whereas there was no significant difference observed between first and second trimester in the albumin: globulin ratio. This may be probably due to imbalance between albumin and globulin concentration which has been earlier attributed to increased plasma volume and synthesis of pregnancy induced plasma proteins.

In conclusion, alteration in function of any sites will affect the appropriate serum protein fractions. Changes in serum protein during normal pregnancy can also be attributed to varying factors like increase in plasma volume and wide individual physiological variations at any given stage of pregnancy. However, a considerable amount of albumin passes through the glomerular filtrate daily during pregnancy and most of this is reabsorbed in the renal tubules, being broken down in the process and are lost to the body. It is therefore imperative to have a protein profile analysis during pregnancy.
Conflict of Interest: None